Modulation of DRAK2 expression

ABSTRACT

Compounds, compositions and methods are provided for modulating the expression of DRAK2. The compositions comprise oligonucleotides, targeted to nucleic acid encoding DRAK2. Methods of using these compounds for modulation of DRAK2 expression and for diagnosis and treatment of disease associated with expression of DRAK2 are provided.

FIELD OF THE INVENTION

[0001] The present invention provides compositions and methods for modulating the expression of DRAK2. In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding DRAK2. Such compounds are shown herein to modulate the expression of DRAK2.

BACKGROUND OF THE INVENTION

[0002] Apoptosis, or programmed cell death, is a naturally occurring process that has been strongly conserved during evolution to prevent uncontrolled cell proliferation. This form of cell suicide plays a crucial role in ensuring the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. However, if this process becomes overstimulated, cell loss and degenerative disorders including neurological disorders such as Alzheimers, Parkinsons, ALS, retinitis pigmentosa and blood cell disorders can result. Stimuli which can trigger apoptosis include growth factors such as tumor necrosis factor (TNF), Fas and transforming growth factor beta (TGFβ), neurotransmitters, growth factor withdrawal, loss of extracellular matrix attachment and extreme fluctuations in intracellular calcium levels (Afford and Randhawa, Mol. Pathol., 2000, 53, 55-63).

[0003] Alternatively, insufficient apoptosis, triggered by growth factors, extracellular matrix changes, CD40 ligand, viral gene products neutral amino acids, zinc, estrogen and androgens, can contribute to the development of cancer, autoimmune disorders and viral infections (Afford and Randhawa, Mol. Pathol., 2000, 53, 55-63). Consequently, apoptosis is regulated under normal circumstances by the interaction of gene products that either induce or inhibit cell death and several gene products which modulate the apoptotic process have now been identified.

[0004] Protein kinases play critical roles in signal transduction in response to a number of external stimuli. Death-associated protein kinase-related apoptosis-inducing protein kinase 2 (DRAK2, also known as serine/threonine kinase 17B (apoptosis inducing); STK17B) is a recently cloned kinase whose catalytic domain is related to that of death-associated protein kinase (DAP), a serine threonine kinase involved in apoptotic signaling by interferon-gamma (Sanjo et al., J. Biol. Chem., 1998, 273, 29066-29071). The full-length DRAK2 cDNA encodes a deduced 42.34 kDa, 372-amino acid protein. The putative kinase domain is located at the amino terminus and contains all 11 subdomains conserved among serine/threonine kinases (Sanjo et al., J. Biol. Chem., 1998, 273, 29066-29071). DRAK2 is expressed in various tissues, such as heart, placenta, liver, and pancreas, as different sized transcripts, presumably due to differences in the 3-prime untranslated region (Sanjo et al., J. Biol. Chem., 1998, 273, 29066-29071).

[0005] The role for DRAK2 in apoptosis signaling has been confirmed by the observation that overexpression of DRAK1 induces morphological changes of apoptosis in NIH 3T3 cells (Sanjo et al., J. Biol. Chem., 1998, 273, 29066-29071).

[0006] Disclosed and claimed in PCT publication WO 99/33961 are nucleic acid sequences encoding DRAK2, said nucleic acid sequences being capable of providing drugs useful in preventing and treating diseases in association with apoptosis. Additionally claimed in this same PCT publication is a nucleic acid consisting of at least 12 bases of the nucleic acid encoding DRAK2 or a complementary strand or derivative thereof, and an antibody capable of binding to DRAK1 (Akira et al., 1999).

[0007] There exists a need to identify methods of modulating apoptosis for the therapeutic treatment of human diseases and it is believed that agents capable of modulating the expression of kinases involved in apoptosis signaling cascades will be integral to these methods.

[0008] Currently, there are no known therapeutic agents which effectively inhibit the synthesis of DRAK2. To date, investigative strategies aimed at modulating DRAK2 function have been limited to the use of antibodies. Consequently, there remains a long felt need for agents capable of effectively inhibiting DRAK2 function.

[0009] Antisense technology is emerging as an effective means for reducing the expression of specific gene products and may therefore prove to be uniquely useful in a number of therapeutic, diagnostic, and research applications for the modulation of gene expression and cellular processes.

[0010] The present invention provides compositions and methods for modulating DRAK2 expression.

SUMMARY OF THE INVENTION

[0011] The present invention is directed to compounds, especially nucleic acid and nucleic acid-like oligomers, which are targeted to a nucleic acid encoding DRAK2, and which modulate the expression of DRAK2. Pharmaceutical and other compositions comprising the compounds of the invention are also provided. Further provided are methods of screening for modulators of DRAK2 and methods of modulating the expression of DRAK2 in cells, tissues or animals comprising contacting said cells, tissues or animals with one or more of the compounds or compositions of the invention. Methods of treating an animal, particularly a human, suspected of having or being prone to a disease or condition associated with expression of DRAK2 are also set forth herein. Such methods comprise administering a therapeutically or prophylactically effective amount of one or more of the compounds or compositions of the invention to the person in need of treatment.

DETAILED DESCRIPTION OF THE INVENTION

[0012] A. Overview of the Invention

[0013] The present invention employs compounds, preferably oligonucleotides and similar species for use in modulating the function or effect of nucleic acid molecules encoding DRAK2. This is accomplished by providing oligonucleotides which specifically hybridize with one or more nucleic acid molecules encoding DRAK2. As used herein, the terms “target nucleic acid” and “nucleic acid molecule encoding DRAK2” have been used for convenience to encompass DNA encoding DRAK2, RNA (including pre-mRNA and mRNA or portions thereof) transcribed from such DNA, and also cDNA derived from such RNA. The hybridization of a compound of this invention with its target nucleic acid is generally referred to as “antisense”. Consequently, the preferred mechanism believed to be included in the practice of some preferred embodiments of the invention is referred to herein as “antisense inhibition.” Such antisense inhibition is typically based upon hydrogen bonding-based hybridization of oligonucleotide strands or segments such that at least one strand or segment is cleaved, degraded, or otherwise rendered inoperable. In this regard, it is presently preferred to target specific nucleic acid molecules and their functions for such antisense inhibition.

[0014] The functions of DNA to be interfered with can include replication and transcription. Replication and transcription, for example, can be from an endogenous cellular template, a vector, a plasmid construct or otherwise. The functions of RNA to be interfered with can include functions such as translocation of the RNA to a site of protein translation, translocation of the RNA to sites within the cell which are distant from the site of RNA synthesis, translation of protein from the RNA, splicing of the RNA to yield one or more RNA species, and catalytic activity or complex formation involving the RNA which may be engaged in or facilitated by the RNA. One preferred result of such interference with target nucleic acid function is modulation of the expression of DRAK2. In the context of the present invention, “modulation” and “modulation of expression” mean either an increase (stimulation) or a decrease (inhibition) in the amount or levels of a nucleic acid molecule encoding the gene, e.g., DNA or RNA. Inhibition is often the preferred form of modulation of expression and mRNA is often a preferred target nucleic acid.

[0015] In the context of this invention, “hybridization” means the pairing of complementary strands of oligomeric compounds. In the present invention, the preferred mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleoside or nucleotide bases (nucleobases) of the strands of oligomeric compounds. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. Hybridization can occur under varying circumstances.

[0016] An antisense compound is specifically hybridizable when binding of the compound to the target nucleic acid interferes with the normal function of the target nucleic acid to cause a loss of activity, and there is a sufficient degree of complementarity to avoid non-specific binding of the antisense compound to non-target nucleic acid sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, and under conditions in which assays are performed in the case of in vitro assays.

[0017] In the present invention the phrase “stringent hybridization conditions” or “stringent conditions” refers to conditions under which a compound of the invention will hybridize to its target sequence, but to a minimal number of other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances and in the context of this invention, “stringent conditions” under which oligomeric compounds hybridize to a target sequence are determined by the nature and composition of the oligomeric compounds and the assays in which they are being investigated.

[0018] “Complementary,” as used herein, refers to the capacity for precise pairing between two nucleobases of an oligomeric compound. For example, if a nucleobase at a certain position of an oligonucleotide (an oligomeric compound), is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, said target nucleic acid being a DNA, RNA, or oligonucleotide molecule, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be a complementary position. The oligonucleotide and the further DNA, RNA, or oligonucleotide molecule are complementary to each other when a sufficient number of complementary positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of precise pairing or complementarity over a sufficient number of nucleobases such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid.

[0019] It is understood in the art that the sequence of an antisense compound need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, an oligonucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). It is preferred that the antisense compounds of the present invention comprise at least 70% sequence complementarity to a target region within the target nucleic acid, more preferably that they comprise 90% sequence complementarity and even more preferably comprise 95% sequence complementarity to the target region within the target nucleic acid sequence to which they are targeted. For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having 4 (four) noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).

[0020] B. Compounds of the Invention

[0021] According to the present invention, compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other oligomeric compounds which hybridize to at least a portion of the target nucleic acid. As such, these compounds may be introduced in the form of single-stranded, double-stranded, circular or hairpin oligomeric compounds and may contain structural elements such as internal or terminal bulges or loops. Once introduced to a system, the compounds of the invention may elicit the action of one or more enzymes or structural proteins to effect modification of the target nucleic acid. One non-limiting example of such an enzyme is RNAse H, a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNAse H. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. Similar roles have been postulated for other ribonucleases such as those in the RNase III and ribonuclease L family of enzymes.

[0022] While the preferred form of antisense compound is a single-stranded antisense oligonucleotide, in many species the introduction of double-stranded structures, such as double-stranded RNA (dsRNA) molecules, has been shown to induce potent and specific antisense-mediated reduction of the function of a gene or its associated gene products. This phenomenon occurs in both plants and animals and is believed to have an evolutionary connection to viral defense and transposon silencing.

[0023] The first evidence that dsRNA could lead to gene silencing in animals came in 1995 from work in the nematode, Caenorhabditis elegans (Guo and Kempheus, Cell, 1995, 81, 611-620). Montgomery et al. have shown that the primary interference effects of dsRNA are posttranscriptional (Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507). The posttranscriptional antisense mechanism defined in Caenorhabditis elegans resulting from exposure to double-stranded RNA (dsRNA) has since been designated RNA interference (RNAi). This term has been generalized to mean antisense-mediated gene silencing involving the introduction of dsRNA leading to the sequence-specific reduction of endogenous targeted mRNA levels (Fire et al., Nature, 1998, 391, 806-811). Recently, it has been shown that it is, in fact, the single-stranded RNA oligomers of antisense polarity of the dsRNAs which are the potent inducers of RNAi (Tijsterman et al., Science, 2002, 295, 694-697).

[0024] In the context of this invention, the term “oligomeric compound” refers to a polymer or oligomer comprising a plurality of monomeric units. In the context of this invention, the term “oligonucleotide” refers to an oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or mimetics, chimeras, analogs and homologs thereof. This term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent internucleoside (backbone) linkages as well as oligonucleotides having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for a target nucleic acid and increased stability in the presence of nucleases.

[0025] While oligonucleotides are a preferred form of the compounds of this invention, the present invention comprehends other families of compounds as well, including but not limited to oligonucleotide analogs and mimetics such as those described herein.

[0026] The compounds in accordance with this invention preferably comprise from about 8 to about 80 nucleobases (i.e. from about 8 to about 80 linked nucleosides). One of ordinary skill in the art will appreciate that the invention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

[0027] In one preferred embodiment, the compounds of the invention are 12 to 50 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases in length.

[0028] In another preferred embodiment, the compounds of the invention are 15 to 30 nucleobases in length. One having ordinary skill in the art will appreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

[0029] Particularly preferred compounds are oligonucleotides from about 12 to about 50 nucleobases, even more preferably those comprising from about 15 to about 30 nucleobases.

[0030] Antisense compounds 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative antisense compounds are considered to be suitable antisense compounds as well.

[0031] Exemplary preferred antisense compounds include oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately upstream of the 5′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). Similarly preferred antisense compounds are represented by oligonucleotide sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred antisense compounds (the remaining nucleobases being a consecutive stretch of the same oligonucleotide beginning immediately downstream of the 3′-terminus of the antisense compound which is specifically hybridizable to the target nucleic acid and continuing until the oligonucleotide contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred antisense compounds illustrated herein will be able, without undue experimentation, to identify further preferred antisense compounds.

[0032] C. Targets of the Invention

[0033] “Targeting” an antisense compound to a particular nucleic acid molecule, in the context of this invention, can be a multistep process. The process usually begins with the identification of a target nucleic acid whose function is to be modulated. This target nucleic acid may be, for example, a cellular gene (or mRNA transcribed from the gene) whose expression is associated with a particular disorder or disease state, or a nucleic acid molecule from an infectious agent. In the present invention, the target nucleic acid encodes DRAK2.

[0034] The targeting process usually also includes determination of at least one target region, segment, or site within the target nucleic acid for the antisense interaction to occur such that the desired effect, e.g., modulation of expression, will result. Within the context of the present invention, the term “region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic. Within regions of target nucleic acids are segments. “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid. “Sites,” as used in the present invention, are defined as positions within a target nucleic acid.

[0035] Since, as is known in the art, the translation initiation codon is typically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in the corresponding DNA molecule), the translation initiation codon is also referred to as the “AUG codon,” the “start codon” or the “AUG start codon”. A minority of genes have a translation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function in vivo. Thus, the terms “translation initiation codon” and “start codon” can encompass many codon sequences, even though the initiator amino acid in each instance is typically methionine (in eukaryotes) or formylmethionine (in prokaryotes). It is also known in the art that eukaryotic and prokaryotic genes may have two or more alternative start codons, any one of which may be preferentially utilized for translation initiation in a particular cell type or tissue, or under a particular set of conditions. In the context of the invention, “start codon” and “translation initiation codon” refer to the codon or codons that are used in vivo to initiate translation of an mRNA transcribed from a gene encoding DRAK2, regardless of the sequence(s) of such codons. It is also known in the art that a translation termination codon (or “stop codon”) of a gene may have one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

[0036] The terms “start codon region” and “translation initiation codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation initiation codon. Similarly, the terms “stop codon region” and “translation termination codon region” refer to a portion of such an mRNA or gene that encompasses from about 25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from a translation termination codon. Consequently, the “start codon region” (or “translation initiation codon region”) and the “stop codon region” (or “translation termination codon region”) are all regions which may be targeted effectively with the antisense compounds of the present invention.

[0037] The open reading frame (ORF) or “coding region,” which is known in the art to refer to the region between the translation initiation codon and the translation termination codon, is also a region which may be targeted effectively. Within the context of the present invention, a preferred region is the intragenic region encompassing the translation initiation or termination codon of the open reading frame (ORF) of a gene.

[0038] Other target regions include the 5′ untranslated region (5′UTR), known in the art to refer to the portion of an mRNA in the 5′ direction from the translation initiation codon, and thus including nucleotides between the 5′ cap site and the translation initiation codon of an mRNA (or corresponding nucleotides on the gene), and the 3′ untranslated region (3′UTR), known in the art to refer to the portion of an mRNA in the 3′ direction from the translation termination codon, and thus including nucleotides between the translation termination codon and 3′ end of an mRNA (or corresponding nucleotides on the gene). The 5′ cap site of an mRNA comprises an N7-methylated guanosine residue joined to the 5′-most residue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap region of an mRNA is considered to include the 5′ cap structure itself as well as the first 50 nucleotides adjacent to the cap site. It is also preferred to target the 5′ cap region.

[0039] Although some eukaryotic mRNA transcripts are directly translated, many contain one or more regions, known as “introns,” which are excised from a transcript before it is translated. The remaining (and therefore translated) regions are known as “exons” and are spliced together to form a continuous mRNA sequence. Targeting splice sites, i.e., intron-exon junctions or exon-intron junctions, may also be particularly useful in situations where aberrant splicing is implicated in disease, or where an overproduction of a particular splice product is implicated in disease. Aberrant fusion junctions due to rearrangements or deletions are also preferred target sites. mRNA transcripts produced via the process of splicing of two (or more) mRNAs from different gene sources are known as “fusion transcripts”. It is also known that introns can be effectively targeted using antisense compounds targeted to, for example, DNA or pre-mRNA.

[0040] It is also known in the art that alternative RNA transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as “variants”. More specifically, “pre-mRNA variants” are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence.

[0041] Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller “mRNA variants”. Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as “alternative splice variants”. If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

[0042] It is also known in the art that variants can be produced through the use of alternative signals to start or stop transcription and that pre-mRNAs and mRNAs can possess more that one start codon- or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as “alternative start variants” of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as “alternative stop variants” of that pre-mRNA or mRNA. One specific type of alternative stop variant is the “polyA variant” in which the multiple transcripts produced result from the alternative selection of one of the “polyA stop signals” by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites. Within the context of the invention, the types of variants described herein are also preferred target nucleic acids.

[0043] The locations on the target nucleic acid to which the preferred antisense compounds hybridize are hereinbelow referred to as “preferred target segments.” As used herein the term “preferred target segment” is defined as at least an 8-nucleobase portion of a target region to which an active antisense compound is targeted. While not wishing to be bound by theory, it is presently believed that these target segments represent portions of the target nucleic acid which are accessible for hybridization.

[0044] While the specific sequences of certain preferred target segments are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional preferred target segments may be identified by one having ordinary skill.

[0045] Target segments 8-80 nucleobases in length comprising a stretch of at least eight (8) consecutive nucleobases selected from within the illustrative preferred target segments are considered to be suitable for targeting as well.

[0046] Target segments can include DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 5′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately upstream of the 5′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). Similarly preferred target segments are represented by DNA or RNA sequences that comprise at least the 8 consecutive nucleobases from the 3′-terminus of one of the illustrative preferred target segments (the remaining nucleobases being a consecutive stretch of the same DNA or RNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the DNA or RNA contains about 8 to about 80 nucleobases). One having skill in the art armed with the preferred target segments illustrated herein will be able, without undue experimentation, to identify further preferred target segments.

[0047] Once one or more target regions, segments or sites have been identified, antisense compounds are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect.

[0048] D. Screening and Target Validation

[0049] In a further embodiment, the “preferred target segments” identified herein may be employed in a screen for additional compounds that modulate the expression of DRAK2. “Modulators” are those compounds that decrease or increase the expression of a nucleic acid molecule encoding DRAK2 and which comprise at least an 8-nucleobase portion which is complementary to a preferred target segment. The screening method comprises the steps of contacting a preferred target segment of a nucleic acid molecule encoding DRAK2 with one or more candidate modulators, and selecting for one or more candidate modulators which decrease or increase the expression of a nucleic acid molecule encoding DRAK2. Once it is shown that the candidate modulator or modulators are capable of modulating (e.g. either decreasing or increasing) the expression of a nucleic acid molecule encoding DRAK2, the modulator may then be employed in further investigative studies of the function of DRAK2, or for use as a research, diagnostic, or therapeutic agent in accordance with the present invention.

[0050] The preferred target segments of the present invention may be also be combined with their respective complementary antisense compounds of the present invention to form stabilized double-stranded (duplexed) oligonucleotides.

[0051] Such double stranded oligonucleotide moieties have been shown in the art to modulate target expression and regulate translation as well as RNA processsing via an antisense mechanism. Moreover, the double-stranded moieties may be subject to chemical modifications (Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature 1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl. Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev., 1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498; Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such double-stranded moieties have been shown to inhibit the target by the classical hybridization of antisense strand of the duplex to the target, thereby triggering enzymatic degradation of the target (Tijsterman et al., Science, 2002, 295, 694-697).

[0052] The compounds of the present invention can also be applied in the areas of drug discovery and target validation. The present invention comprehends the use of the compounds and preferred target segments identified herein in drug discovery efforts to elucidate relationships that exist between DRAK2 and a disease state, phenotype, or condition. These methods include detecting or modulating DRAK2 comprising contacting a sample, tissue, cell, or organism with the compounds of the present invention, measuring the nucleic acid or protein level of DRAK2 and/or a related phenotypic or chemical endpoint at some time after treatment, and optionally comparing the measured value to a non-treated sample or sample treated with a further compound of the invention. These methods can also be performed in parallel or in combination with other experiments to determine the function of unknown genes for the process of target validation or to determine the validity of a particular gene product as a target for treatment or prevention of a particular disease, condition, or phenotype.

[0053] E. Kits, Research Reagents, Diagnostics, and Therapeutics

[0054] The compounds of the present invention can be utilized for diagnostics, therapeutics, prophylaxis and as research reagents and kits. Furthermore, antisense oligonucleotides, which are able to inhibit gene expression with exquisite specificity, are often used by those of ordinary skill to elucidate the function of particular genes or to distinguish between functions of various members of a biological pathway.

[0055] For use in kits and diagnostics, the compounds of the present invention, either alone or in combination with other compounds or therapeutics, can be used as tools in differential and/or combinatorial analyses to elucidate expression patterns of a portion or the entire complement of genes expressed within cells and tissues.

[0056] As one nonlimiting example, expression patterns within cells or tissues treated with one or more antisense compounds are compared to control cells or tissues not treated with antisense compounds and the patterns produced are analyzed for differential levels of gene expression as they pertain, for example, to disease association, signaling pathway, cellular localization, expression level, size, structure or function of the genes examined. These analyses can be performed on stimulated or unstimulated cells and in the presence or absence of other compounds which affect expression patterns.

[0057] Examples of methods of gene expression analysis known in the art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett., 2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE (serial analysis of gene expression)(Madden, et al., Drug Discov. Today, 2000, 5, 415-425), READS (restriction enzyme amplification of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999, 303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16; Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000, 480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57), subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal. Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41, 203-208), subtractive cloning, differential display (DD) (Jurecic and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl., 1998, 31, 286-96), FISH (fluorescent in situ hybridization) techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35, 1895-904) and mass spectrometry methods (To, Comb. Chem. High Throughput Screen, 2000, 3, 235-41).

[0058] The compounds of the invention are useful for research and diagnostics, because these compounds hybridize to nucleic acids encoding DRAK2. For example, oligonucleotides that are shown to hybridize with such efficiency and under such conditions as disclosed herein as to be effective DRAK2 inhibitors will also be effective primers or probes under conditions favoring gene amplification or detection, respectively. These primers and probes are useful in methods requiring the specific detection of nucleic acid molecules encoding DRAK2 and in the amplification of said nucleic acid molecules for detection or for use in further studies of DRAK2. Hybridization of the antisense oligonucleotides, particularly the primers and probes, of the invention with a nucleic acid encoding DRAK2 can be detected by means known in the art. Such means may include conjugation of an enzyme to the oligonucleotide, radiolabelling of the oligonucleotide or any other suitable detection means. Kits using such detection means for detecting the level of DRAK2 in a sample may also be prepared.

[0059] The specificity and sensitivity of antisense is also harnessed by those of skill in the art for therapeutic uses. Antisense compounds have been employed as therapeutic moieties in the treatment of disease states in animals, including humans. Antisense oligonucleotide drugs, including ribozymes, have been safely and effectively administered to humans and numerous clinical trials are presently underway. It is thus established that antisense compounds can be useful therapeutic modalities that can be configured to be useful in treatment regimes for the treatment of cells, tissues and animals, especially humans.

[0060] For therapeutics, an animal, preferably a human, suspected of having a disease or disorder which can be treated by modulating the expression of DRAK2 is treated by administering antisense compounds in accordance with this invention. For example, in one non-limiting embodiment, the methods comprise the step of administering to the animal in need of treatment, a therapeutically effective amount of a DRAK2 inhibitor. The DRAK2 inhibitors of the present invention effectively inhibit the activity of the DRAK2 protein or inhibit the expression of the DRAK2 protein. In one embodiment, the activity or expression of DRAK2 in an animal is inhibited by about 10%. Preferably, the activity or expression of DRAK2 in an animal is inhibited by about 30%. More preferably, the activity or expression of DRAK2 in an animal is inhibited by 50% or more.

[0061] For example, the reduction of the expression of DRAK2 may be measured in serum, adipose tissue, liver or any other body fluid, tissue or organ of the animal. Preferably, the cells contained within said fluids, tissues or organs being analyzed-contain a nucleic acid molecule encoding DRAK2 protein and/or the DRAK2 protein itself.

[0062] The compounds of the invention can be utilized in pharmaceutical compositions by adding an effective amount of a compound to a suitable pharmaceutically acceptable diluent or carrier. Use of the compounds and methods of the invention may also be useful prophylactically.

[0063] F. Modifications

[0064] As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric compound can be further joined to form a circular compound, however, linear compounds are generally preferred. In addition, linear compounds may have internal nucleobase complementarity and may therefore fold in a manner as to produce a fully or partially double-stranded compound. Within oligonucleotides, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

[0065] Modified Internucleoside Linkages (Backbones)

[0066] Specific examples of preferred antisense compounds useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.

[0067] Preferred modified oligonucleotide backbones containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Preferred oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof). Various salts, mixed salts and free acid forms are also included.

[0068] Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0069] Preferred modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

[0070] Representative United States patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain of which are commonly owned with this application, and each of which is herein incorporated by reference.

[0071] Modified Sugar and Internucleoside Linkages—Mimetics

[0072] In other preferred oligonucleotide mimetics, both the sugar and the internucleoside linkage (i.e. the backbone), of the nucleotide units are replaced with novel groups. The nucleobase units are maintained for hybridization with an appropriate target nucleic acid. One such compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0073] Preferred embodiments of the invention are oligonucleotides with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above referenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotides having morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

[0074] Modified Sugars

[0075] Modified oligonucleotides may also contain one or more substituted sugar moieties. Preferred oligonucleotides comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Other preferred oligonucleotides comprise one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred modification includes 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples hereinbelow, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

[0076] Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl (2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be in the arabino (up) position or ribo (down) position. A preferred 2′-arabino modification is 2′-F. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747; and 5,700,920, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0077] A further preferred modification of the sugar includes Locked Nucleic Acids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′ carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety. The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′ oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs and preparation thereof are described in WO 98/39352 and WO 99/14226.

[0078] Natural and Modified Nucleobases

[0079] Oligonucleotides may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡—C—CH₃) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4-]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. T., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

[0080] Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference, and U.S. Pat. No. 5,750,692, which is commonly owned with the instant application and also herein incorporated by reference.

[0081] Conjugates

[0082] Another modification of the oligonucleotides of the invention involves chemically linking to the oligonucleotide one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the oligonucleotide. These moieties or conjugates can include conjugate groups covalently bound to functional groups such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this invention, include groups that improve uptake, enhance resistance to degradation, and/or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application PCT/US92/09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860, the entire disclosure of which are incorporated herein by reference. Conjugate moieties include but are not limited to lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of the invention may also be conjugated to active drug substances, for example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an antibacterial or an antibiotic. Oligonucleotide-drug conjugates and their preparation are described in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15, 1999) which is incorporated herein by reference in its entirety.

[0083] Representative United States patents that teach the preparation of such oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference.

[0084] Chimeric Compounds

[0085] It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single compound or even at a single nucleoside within an oligonucleotide.

[0086] The present invention also includes antisense compounds which are chimeric compounds. “Chimeric” antisense compounds or “chimeras,” in the context of this invention, are antisense compounds, particularly oligonucleotides, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide compound. These oligonucleotides typically contain at least one region wherein the oligonucleotide is modified so as to confer upon the oligonucleotide increased resistance to nuclease degradation, increased cellular uptake, increased stability and/or increased binding affinity for the target nucleic acid. An additional region of the oligonucleotide may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAse H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide-mediated inhibition of gene expression. The cleavage of RNA:RNA hybrids can, in like fashion, be accomplished through the actions of endoribonucleases, such as RNAseL which cleaves both cellular and viral RNA. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

[0087] Chimeric antisense compounds of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, certain of which are commonly owned with the instant application, and each of which is herein incorporated by reference in its entirety.

[0088] G. Formulations

[0089] The compounds of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor-targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption-assisting formulations include, but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

[0090] The antisense compounds of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound which, upon administration to an animal, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.

[0091] The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention are prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives according to the methods disclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

[0092] The term “pharmaceutically acceptable salts” refers to physiologically and pharmaceutically acceptable salts of the compounds of the invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. For oligonucleotides, preferred examples of pharmaceutically acceptable salts and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0093] The present invention also includes pharmaceutical compositions and formulations which include the antisense compounds of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Oligonucleotides with at least one 2′-O-methoxyethyl modification are believed to be particularly useful for oral administration. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

[0094] The pharmaceutical formulations of the present invention, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0095] The compositions of the present invention may be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention may also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions may further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0096] Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, foams and liposome-containing formulations. The pharmaceutical compositions and formulations of the present invention may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients.

[0097] Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Microemulsions are included as an embodiment of the present invention. Emulsions and their uses are well known in the art and are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0098] Formulations of the present invention include liposomal formulations. As used in the present invention, the term “liposome” means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior that contains the composition to be delivered. Cationic liposomes are positively charged liposomes which are believed to interact with negatively charged DNA molecules to form a stable complex. Liposomes that are pH-sensitive or negatively-charged are believed to entrap DNA rather than complex with it. Both cationic and noncationic liposomes have been used to deliver DNA to cells.

[0099] Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. Liposomes and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0100] The pharmaceutical formulations and compositions of the present invention may also include surfactants. The use of surfactants in drug products, formulations and in emulsions is well known in the art. Surfactants and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0101] In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly oligonucleotides. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs. Penetration enhancers may be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants. Penetration enhancers and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety.

[0102] One of skill in the art will recognize that formulations are routinely designed according to their intended use, i.e. route of administration.

[0103] Preferred formulations for topical administration include those in which the oligonucleotides of the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Preferred lipids and liposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g. dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA).

[0104] For topical or other administration, oligonucleotides of the invention may be encapsulated within liposomes or may form complexes thereto, in particular to cationic liposomes. Alternatively, oligonucleotides may be complexed to lipids, in particular to cationic lipids. Preferred fatty acids and esters, pharmaceutically acceptable salts thereof, and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Topical formulations are described in detail in U.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, which is incorporated herein by reference in its entirety.

[0105] Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable. Preferred oral formulations are those in which oligonucleotides of the invention are administered in conjunction with one or more penetration enhancers surfactants and chelators. Preferred surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Preferred bile acids/salts and fatty acids and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Also preferred are combinations of penetration enhancers, for example, fatty acids/salts in combination with bile acids/salts. A particularly preferred combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention may be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. Oligonucleotide complexing agents and their uses are further described in U.S. Pat. No. 6,287,860, which is incorporated herein in its entirety. Oral formulations for oligonucleotides and their preparation are described in detail in U.S. application Ser. No. 09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20, 1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is incorporated herein by reference in their entirety.

[0106] Compositions and formulations for parenteral, intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

[0107] Certain embodiments of the invention provide pharmaceutical compositions containing one or more oligomeric compounds and one or more other chemotherapeutic agents which function by a non-antisense mechanism. Examples of such chemotherapeutic agents include but are not limited to cancer chemotherapeutic drugs such as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU) 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES). When used with the compounds of the invention, such chemotherapeutic agents may be used individually (e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for a period of time followed by MTX and oligonucleotide), or in combination with one or more other such chemotherapeutic agents (e.g., 5′-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs, including but not limited to nonsteroidal anti-inflammatory drugs and corticosteroids, and antiviral drugs, including but not limited to ribivirin, vidarabine, acyclovir and ganciclovir, may also be combined in compositions of the invention. Combinations of antisense compounds and other non-antisense drugs are also within the scope of this invention. Two or more combined compounds may be used together or sequentially.

[0108] In another related embodiment, compositions of the invention may contain one or more antisense compounds, particularly oligonucleotides, targeted to a first nucleic acid and one or more additional antisense compounds targeted to a second nucleic acid target. Alternatively, compositions of the invention may contain two or more antisense compounds targeted to different regions of the same nucleic acid target. Numerous examples of antisense compounds are known in the art. Two or more combined compounds may be used together or sequentially.

[0109] H. Dosing

[0110] The formulation of therapeutic compositions and their subsequent administration (dosing) is believed to be within the skill of those in the art. Dosing is dependent on severity and responsiveness of the disease state to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC₅₀s found to be effective in in vitro and in vivo animal models. In general, dosage is from 0.01 ug to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly, or even once every 2 to 20 years. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 ug to 100 g per kg of body weight, once or more daily, to once every 20 years.

[0111] While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same.

EXAMPLES Example 1

[0112] Synthesis of Nucleoside Phosphoramidites

[0113] The following compounds, including amidites and their intermediates were prepared as described in U.S. Pat. No. 6,426,220 and published PCT WO 02/36743; 5′-O-Dimethoxytrityl-thymidine intermediate for 5-methyl dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for 5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N-4-benzoyl-5-methylcytidine penultimate intermediate for 5-methyl dC amidite, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N-4-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine, 2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modified amidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate, 5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T amidite), 5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidine intermediate, 5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N-4-benzoyl-5-methyl-cytidine penultimate intermediate, [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE A amdite), [5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites, 2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine, 2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine, 5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine, 5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N dimethylaminooxyethyl]-5-methyluridine, 2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine, 5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-(Aminooxyethoxy) nucleoside amidites, N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite], 2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites, 2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine, 5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2

[0114] Oligonucleotide and Oligonucleoside Synthesis

[0115] The antisense compounds used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.

[0116] Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O) oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry with oxidation by iodine.

[0117] Phosphorothioates (P═S) are synthesized similar to phosphodiester oligonucleotides with the following exceptions: thiation was effected by utilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the oxidation of the phosphite linkages. The thiation reaction step time was increased to 180 sec and preceded by the normal capping step. After cleavage from the CPG column and deblocking in concentrated ammonium hydroxide at 55° C. (12-16 hr), the oligonucleotides were recovered by precipitating with >3 volumes of ethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides are prepared as described in U.S. Pat. No. 5,508,270, herein incorporated by reference.

[0118] Alkyl phosphonate oligonucleotides are prepared as described in U.S. Pat. No. 4,469,863, herein incorporated by reference.

[0119] 3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein incorporated by reference.

[0120] Phosphoramidite oligonucleotides are prepared as described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated by reference.

[0121] Alkylphosphonothioate oligonucleotides are prepared as described in published PCT applications PCT/US94/00902 and PCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively), herein incorporated by reference.

[0122] 3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared as described in U.S. Pat. No. 5,476,925, herein incorporated by reference.

[0123] Phosphotriester oligonucleotides are prepared as described in U.S. Pat. No. 5,023,243, herein incorporated by reference.

[0124] Borano phosphate oligonucleotides are prepared as described in U.S. Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

[0125] Oligonucleosides: Methylenemethylimino linked oligonucleosides, also identified as MMI linked oligonucleosides, methylenedimethylhydrazo linked oligonucleosides, also identified as MDH linked oligonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified as amide-3 linked oligonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified as amide-4 linked oligonucleosides, as well as mixed backbone compounds having, for instance, alternating MMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of which are herein incorporated by reference.

[0126] Formacetal and thioformacetal linked oligonucleosides are prepared as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporated by reference.

[0127] Ethylene oxide linked oligonucleosides are prepared as described in U.S. Pat. No. 5,223,618, herein incorporated by reference.

Example 3

[0128] RNA Synthesis

[0129] In general, RNA synthesis chemistry is based on the selective incorporation of various protecting groups at strategic intermediary reactions. Although one of ordinary skill in the art will understand the use of protecting groups in organic synthesis, a useful class of protecting groups includes silyl ethers. In particular bulky silyl ethers are used to protect the 5′-hydroxyl in combination with an acid-labile orthoester protecting group on the 2′-hydroxyl. This set of protecting groups is then used with standard solid-phase synthesis technology. It is important to lastly remove the acid labile orthoester protecting group after all other synthetic steps. Moreover, the early use of the silyl protecting groups during synthesis ensures facile removal when desired, without undesired deprotection of 2′ hydroxyl.

[0130] Following this procedure for the sequential protection of the 5′-hydroxyl in combination with protection of the 2′-hydroxyl by protecting groups that are differentially removed and are differentially chemically labile, RNA oligonucleotides were synthesized.

[0131] RNA oligonucleotides are synthesized in a stepwise fashion. Each nucleotide is added sequentially (3′- to 5′-direction) to a solid support-bound oligonucleotide. The first nucleoside at the 3′-end of the chain is covalently attached to a solid support. The nucleotide precursor, a ribonucleoside phosphoramidite, and activator are added, coupling the second base onto the 5′-end of the first nucleoside. The support is washed and any unreacted 5′-hydroxyl groups are capped with acetic anhydride to yield 5′-acetyl moieties. The linkage is then oxidized to the more stable and ultimately desired P(V) linkage. At the end of the nucleotide addition cycle, the 5′-silyl group is cleaved with fluoride. The cycle is repeated for each subsequent nucleotide.

[0132] Following synthesis, the methyl protecting groups on the phosphates are cleaved in 30 minutes utilizing 1 M disodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂) in DMF. The deprotection solution is washed from the solid support-bound oligonucleotide using water. The support is then treated with 40% methylamine in water for 10 minutes at 55° C. This releases the RNA oligonucleotides into solution, deprotects the exocyclic amines, and modifies the 2′-groups. The oligonucleotides can be analyzed by anion exchange HPLC at this stage.

[0133] The 2′-orthoester groups are the last protecting groups to be removed. The ethylene glycol monoacetate orthoester protecting group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is one example of a useful orthoester protecting group which, has the following important properties. It is stable to the conditions of nucleoside phosphoramidite synthesis and oligonucleotide synthesis. However, after oligonucleotide synthesis the oligonucleotide is treated with methylamine which not only cleaves the oligonucleotide from the solid support but also removes the acetyl groups from the orthoesters. The resulting 2-ethylhydroxyl substituents on the orthoester are less electron withdrawing than the acetylated precursor. As a result, the modified orthoester becomes more labile to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is approximately 10 times faster after the acetyl groups are removed. Therefore, this orthoester possesses sufficient stability in order to be compatible with oligonucleotide synthesis and yet, when subsequently modified, permits deprotection to be carried out under relatively mild aqueous conditions compatible with the final RNA oligonucleotide product.

[0134] Additionally, methods of RNA synthesis are well known in the art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996; Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821; Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103, 3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett., 1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand., 1990, 44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25, 4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23, 2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23, 2315-2331).

[0135] RNA antisense compounds (RNA oligonucleotides) of the present invention can be synthesized by the methods herein or purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once synthesized, complementary RNA antisense compounds can then be annealed by methods known in the art to form double stranded (duplexed) antisense compounds. For example, duplexes can be formed by combining 30 μl of each of the complementary strands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and 15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90° C., then 1 hour at 37° C. The resulting duplexed antisense compounds can be used in kits, assays, screens, or other methods to investigate the role of a target nucleic acid.

Example 4

[0136] Synthesis of Chimeric Oligonucleotides

[0137] Chimeric oligonucleotides, oligonucleosides or mixed oligonucleotides/oligonucleosides of the invention can be of several different types. These include a first type wherein the “gap” segment of linked nucleosides is positioned between 5′ and 3′ “wing” segments of linked nucleosides and a second “open end” type wherein the “gap” segment is located at either the 3′ or the 5′ terminus of the oligomeric compound. Oligonucleotides of the first type are also known in the art as “gapmers” or gapped oligonucleotides. Oligonucleotides of the second type are also known in the art as “hemimers” or “wingmers”.

[0138] [2′-O-Me]—[2′-deoxy]—[2′-O-Me] Chimeric Phosphorothioate Oligonucleotides

[0139] Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and 2′-deoxy phosphorothioate oligonucleotide segments are synthesized using an Applied Biosystems automated DNA synthesizer Model 394, as above. Oligonucleotides are synthesized using the automated synthesizer and 2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings. The standard synthesis cycle is modified by incorporating coupling steps with increased reaction times for the 5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protected oligonucleotide is cleaved from the support and deprotected in concentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotected oligo is then recovered by an appropriate method (precipitation, column chromatography, volume reduced in vacuo and analyzed spetrophotometrically for yield and for purity by capillary electrophoresis and by mass spectrometry.

[0140] [2′-O-(2-Methoxyethyl)]—[2′-deoxy]—[2′-O-(Methoxyethyl)] Chimeric Phosphorothioate Oligonucleotides

[0141] [2′-O-(2-methoxyethyl)]—[2′-deoxy]—[2′-O (methoxyethyl)] chimeric phosphorothioate oligonucleotides were prepared as per the procedure above for the 2′-O-methyl chimeric oligonucleotide, with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites.

[0142] [2′-O-(2-Methoxyethyl)Phosphodiester]—[2′-deoxy Phosphorothioate]—[2′-O-(2-Methoxyethyl)Phosphodiester] Chimeric Oligonucleotides

[0143] [2′-O-(2-methoxyethyl phosphodiester]—[2′-deoxy phosphorothioate]—[2′-O-(methoxyethyl) phosphodiester] chimeric oligonucleotides are prepared as per the above procedure for the 2′-O-methyl chimeric oligonucleotide with the substitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidation with iodine to generate the phosphodiester internucleotide linkages within the wing portions of the chimeric structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate internucleotide linkages for the center gap.

[0144] Other chimeric oligonucleotides, chimeric oligonucleosides and mixed chimeric oligonucleotides/oligonucleosides are synthesized according to U.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5

[0145] Design and Screening of Duplexed Antisense Compounds Targeting DRAK2

[0146] In accordance with the present invention, a series of nucleic acid duplexes comprising the antisense compounds of the present invention and their complements can be designed to target DRAK2. The nucleobase sequence of the antisense strand of the duplex comprises at least a portion of an oligonucleotide in Table 1. The ends of the strands may be modified by the addition of one or more natural or modified nucleobases to form an overhang. The sense strand of the dsRNA is then designed and synthesized as the complement of the antisense strand and may also contain modifications or additions to either terminus. For example, in one embodiment, both strands of the dsRNA duplex would be complementary over the central nucleobases, each having overhangs at one or both termini.

[0147] For example, a duplex comprising an antisense strand having the sequence CGAGAGGCGGACGGGACCG and having a two-nucleobase overhang of deoxythymidine(dT) would have the following structure:   cgagaggcggacgggaccgTT Antisense Strand   ||||||||||||||||||| TTgctctccgcctgccctggc Complement

[0148] RNA strands of the duplex can be synthesized by methods disclosed herein or purchased from Dharmacon Research Inc., (Lafayette, Colo.). Once synthesized, the complementary strands are annealed. The single strands are aliquoted and diluted to a concentration of 50 uM. Once diluted, 30 uL of each strand is combined with 15 uL of a 5× solution of annealing buffer. The final concentration of said buffer is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The final volume is 75 uL. This solution is incubated for 1 minute at 90° C. and then centrifuged for 15 seconds. The tube is allowed to sit for 1 hour at 37° C. at which time the dsRNA duplexes are used in experimentation. The final concentration of the dsRNA duplex is 20 uM. This solution can be stored frozen (−20° C.) and freeze-thawed up to 5 times.

[0149] Once prepared, the duplexed antisense compounds are evaluated for their ability to modulate DRAK2 expression.

[0150] When cells reached 80% confluency, they are treated with duplexed antisense compounds of the invention. For cells grown in 96-well plates, wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (Gibco BRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mL LIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at a final concentration of 200 nM. After 5 hours of treatment, the medium is replaced with fresh medium. Cells are harvested 16 hours after treatment, at which time RNA is isolated and target reduction measured by RT-PCR.

Example 6

[0151] Oligonucleotide Isolation

[0152] After cleavage from the controlled pore glass solid support and deblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours, the oligonucleotides or oligonucleosides are recovered by precipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesized oligonucleotides were analyzed by electrospray mass spectroscopy (molecular weight determination) and by capillary gel electrophoresis and judged to be at least 70% full length material. The relative amounts of phosphorothioate and phosphodiester linkages obtained in the synthesis was determined by the ratio of correct molecular weight relative to the −16 amu product (+/−32+/−48). For some studies oligonucleotides were purified by HPLC, as described by Chiang et al., J. Biol. Chem. 1991, 266, 18162-18171. Results obtained with HPLC-purified material were similar to those obtained with non-HPLC purified material.

Example 7

[0153] Oligonucleotide Synthesis—96 Well Plate Format

[0154] Oligonucleotides were synthesized via solid phase P(III) phosphoramidite chemistry on an automated synthesizer capable of assembling 96 sequences simultaneously in a 96-well format. Phosphodiester internucleotide linkages were afforded by oxidation with aqueous iodine. Phosphorothioate internucleotide linkages were generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were purchased from commercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesized as per standard or patented methods. They are utilized as base protected beta-cyanoethyldiisopropyl phosphoramidites.

[0155] Oligonucleotides were cleaved from support and deprotected with concentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hours and the released product then dried in vacuo. The dried product was then re-suspended in sterile water to afford a master plate from which all analytical and test plate samples are then diluted utilizing robotic pipettors.

Example 8

[0156] Oligonucleotide Analysis—96-Well Plate Format

[0157] The concentration of oligonucleotide in each well was assessed by dilution of samples and UV absorption spectroscopy. The full-length integrity of the individual products was evaluated by capillary electrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ) or, for individually prepared samples, on a commercial CE apparatus (e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition was confirmed by mass analysis of the compounds utilizing electrospray-mass spectroscopy. All assay test plates were diluted from the master plate using single and multi-channel robotic pipettors. Plates were judged to be acceptable if at least 85% of the compounds on the plate were at least 85% full length.

Example 9

[0158] Cell Culture and Oligonucleotide Treatment

[0159] The effect of antisense compounds on target nucleic acid expression can be tested in any of a variety of cell types provided that the target nucleic acid is present at measurable levels. This can be routinely determined using, for example, PCR or Northern blot analysis. The following cell types are provided for illustrative purposes, but other cell types can be routinely used, provided that the target is expressed in the cell type chosen. This can be readily determined by methods routine in the art, for example Northern blot analysis, ribonuclease protection assays, or RT-PCR.

[0160] T-24 cells:

[0161] The human transitional cell bladder carcinoma cell line T-24 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). T-24 cells were routinely cultured in complete McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence. Cells were seeded into 96-well plates (Falcon-Primaria #353872) at a density of 7000 cells/well for use in RT-PCR analysis.

[0162] For Northern blotting or other analysis, cells may be seeded onto 100 mm or other standard tissue culture plates and treated similarly, using appropriate volumes of medium and oligonucleotide.

[0163] A549 Cells:

[0164] The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells were routinely cultured in DMEM basal media (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.). Cells were routinely passaged by trypsinization and dilution when they reached 90% confluence.

[0165] NHDF Cells:

[0166] Human neonatal dermal fibroblast (NHDF) were obtained from the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely maintained in Fibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.) supplemented as recommended by the supplier. Cells were maintained for up to 10 passages as recommended by the supplier.

[0167] HEK Cells:

[0168] Human embryonic keratinocytes (HEK) were obtained from the Clonetics Corporation (Walkersville, Md.). HEKs were routinely maintained in Keratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.) formulated as recommended by the supplier. Cells were routinely maintained for up to 10 passages as recommended by the supplier.

[0169] Treatment with Antisense Compounds:

[0170] When cells reached 65-75% confluency, they were treated with oligonucleotide. For cells grown in 96-well plates, wells were washed once with 100 μL OPTI-MEM™-1 reduced-serum medium (Invitrogen Corporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM™-1 containing 3.75 μg/mL LIPOFECTIN™ (Invitrogen Corporation, Carlsbad, Calif.) and the desired concentration of oligonucleotide. Cells are treated and data are obtained in triplicate. After 4-7 hours of treatment at 37° C., the medium was replaced with fresh medium. Cells were harvested 16-24 hours after oligonucleotide treatment.

[0171] The concentration of oligonucleotide used varies from cell line to cell line. To determine the optimal oligonucleotide concentration for a particular cell line, the cells are treated with a positive control oligonucleotide at a range of concentrations. For human cells the positive control oligonucleotide is selected from either ISIS 13920 (TCCGTCATCGCTCCTCAGGG, SEQ ID NO: 1) which is targeted to human H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO: 2) which is targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are 2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone. For mouse or rat cells the positive control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO: 3, a 2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with a phosphorothioate backbone which is targeted to both mouse and rat c-raf. The concentration of positive control oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is then utilized as the screening concentration for new oligonucleotides in subsequent experiments for that cell line. If 80% inhibition is not achieved, the lowest concentration of positive control oligonucleotide that results in 60% inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide screening concentration in subsequent experiments for that cell line. If 60% inhibition is not achieved, that particular cell line is deemed as unsuitable for oligonucleotide transfection experiments. The concentrations of antisense oligonucleotides used herein are from 50 nM to 300 nM.

Example 10

[0172] Analysis of Oligonucleotide Inhibition of DRAK2 Expression

[0173] Antisense modulation of DRAK2 expression can be assayed in a variety of ways known in the art. For example, DRAK2 mRNA levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitative PCR is presently preferred. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis of the present invention is the use of total cellular RNA as described in other examples herein. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Real-time quantitative (PCR) can be conveniently accomplished using the commercially available ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System, available from PE-Applied Biosystems, Foster City, Calif. and used according to manufacturer's instructions.

[0174] Protein levels of DRAK2 can be quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA) or fluorescence-activated cell sorting (FACS). Antibodies directed to DRAK2 can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art.

Example 11

[0175] Design of Phenotypic Assays and In Vivo Studies for the Use of DRAK2 Inhibitors

[0176] Phenotypic Assays

[0177] Once DRAK2 inhibitors have been identified by the methods disclosed herein, the compounds are further investigated in one or more phenotypic assays, each having measurable endpoints predictive of efficacy in the treatment of a particular disease state or condition. Phenotypic assays, kits and reagents for their use are well known to those skilled in the art and are herein used to investigate the role and/or association of DRAK2 in health and disease. Representative phenotypic assays, which can be purchased from any one of several commercial vendors, include those for determining cell viability, cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston, Mass.), protein-based assays including enzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene Research Products, San Diego, Calif.), cell regulation, signal transduction, inflammation, oxidative processes and apoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube formation assays, cytokine and hormone assays and metabolic assays (Chemicon International Inc., Temecula, Calif.; Amersham Biosciences, Piscataway, N.J.).

[0178] In one non-limiting example, cells determined to be appropriate for a particular phenotypic assay (i.e., MCF-7 cells selected for breast cancer studies; adipocytes for obesity studies) are treated with DRAK2 inhibitors identified from the in vitro studies as well as control compounds at optimal concentrations which are determined by the methods described above. At the end of the treatment period, treated and untreated cells are analyzed by one or more methods specific for the assay to determine phenotypic outcomes and endpoints.

[0179] Phenotypic endpoints include changes in cell morphology over time or treatment dose as well as changes in levels of cellular components such as proteins, lipids, nucleic acids, hormones, saccharides or metals. Measurements of cellular status which include pH, stage of the cell cycle, intake or excretion of biological indicators by the cell, are also endpoints of interest.

[0180] Analysis of the geneotype of the cell (measurement of the expression of one or more of the genes of the cell) after treatment is also used as an indicator of the efficacy or potency of the DRAK2 inhibitors. Hallmark genes, or those genes suspected to be associated with a specific disease state, condition, or phenotype, are measured in both treated and untreated cells.

[0181] In Vivo Studies

[0182] The individual subjects of the in vivo studies described herein are warm-blooded vertebrate animals, which includes humans.

[0183] The clinical trial is subjected to rigorous controls to ensure that individuals are not unnecessarily put at risk and that they are fully informed about their role in the study. To account for the psychological effects of receiving treatments, volunteers are randomly given placebo or DRAK2 inhibitor. Furthermore, to prevent the doctors from being biased in treatments, they are not informed as to whether the medication they are administering is a DRAK2 inhibitor or a placebo. Using this randomization approach, each volunteer has the same chance of being given either the new treatment or the placebo.

[0184] Volunteers receive either the DRAK2 inhibitor or placebo for eight week period with biological parameters associated with the indicated disease state or condition being measured at the beginning (baseline measurements before any treatment), end (after the final treatment), and at regular intervals during the study period. Such measurements include the levels of nucleic acid molecules encoding DRAK2 or DRAK2 protein levels in body fluids, tissues or organs compared to pre-treatment levels. Other measurements include, but are not limited to, indices of the disease state or condition being treated, body weight, blood pressure, serum titers of pharmacologic indicators of disease or toxicity as well as ADME (absorption, distribution, metabolism and excretion) measurements.

[0185] Information recorded for each patient includes age (years), gender, height (cm), family history of disease state or condition (yes/no), motivation rating (some/moderate/great) and number and type of previous treatment regimens for the indicated disease or condition.

[0186] Volunteers taking part in this study are healthy adults (age 18 to 65 years) and roughly an equal number of males and females participate in the study. Volunteers with certain characteristics are equally distributed for placebo and DRAK2 inhibitor treatment. In general, the volunteers treated with placebo have little or no response to treatment, whereas the volunteers treated with the DRAK2 inhibitor show positive trends in their disease state or condition index at the conclusion of the study.

Example 12

[0187] RNA Isolation

[0188] Poly(A)+ mRNA Isolation

[0189] Poly(A)+ mRNA was isolated according to Miura et al., (Clin. Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine in the art. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to each well, the plate was gently agitated and then incubated at room temperature for five minutes. 55 μL of lysate was transferred to Oligo d(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated for 60 minutes at room temperature, washed 3 times with 200 μL of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plate was blotted on paper towels to remove excess wash buffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6), preheated to 70° C., was added to each well, the plate was incubated on a 90° C. hot plate for 5 minutes, and the eluate was then transferred to a fresh 96-well plate.

[0190] Cells grown on 100 mm or other standard plates may be treated similarly, using appropriate volumes of all solutions.

[0191] Total RNA Isolation

[0192] Total RNA was isolated using an RNEASY 96™ kit and buffers purchased from Qiagen Inc. (Valencia, Calif.) following the manufacturer's recommended procedures. Briefly, for cells grown on 96-well plates, growth medium was removed from the cells and each well was washed with 200 μL cold PBS. 150 μL Buffer RLT was added to each well and the plate vigorously agitated for 20 seconds. 150 μL of 70% ethanol was then added to each well and the contents mixed by pipetting three times up and down. The samples were then transferred to the RNEASY 96™ well plate attached to a QIAVAC™ manifold fitted with a waste collection tray and attached to a vacuum source. Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and incubated for 15 minutes and the vacuum was again applied for 1 minute. An additional 500 μL of Buffer RW1 was added to each well of the RNEASY 96™ plate and the vacuum was applied for 2 minutes. 1 mL of Buffer RPE was then added to each well of the RNEASY 96™ plate and the vacuum applied for a period of 90 seconds. The Buffer RPE wash was then repeated and the vacuum was applied for an additional 3 minutes. The plate was then removed from the QIAVAC™ manifold and blotted dry on paper towels. The plate was then re-attached to the QIAVAC™ manifold fitted with a collection tube rack containing 1.2 mL collection tubes. RNA was then eluted by pipetting 140 μL of RNAse free water into each well, incubating 1 minute, and then applying the vacuum for 3 minutes.

[0193] The repetitive pipetting and elution steps may be automated using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially, after lysing of the cells on the culture plate, the plate is transferred to the robot deck where the pipetting, DNase treatment and elution steps are carried out.

Example 13

[0194] Real-Time Quantitative PCR Analysis of DRAK2 mRNA Levels

[0195] Quantitation of DRAK2 mRNA levels was accomplished by real-time quantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.) according to manufacturer's instructions. This is a closed-tube, non-gel-based, fluorescence detection system which allows high-throughput quantitation of polymerase chain reaction (PCR) products in real-time. As opposed to standard PCR in which amplification products are quantitated after the PCR is completed, products in real-time quantitative PCR are quantitated as they accumulate. This is accomplished by including in the PCR reaction an oligonucleotide probe that anneals specifically between the forward and reverse PCR primers, and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 5′ end of the probe and a quencher dye (e.g., TAMRA, obtained from either PE-Applied Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end of the probe. When the probe and dyes are intact, reporter dye emission is quenched by the proximity of the 3′ quencher dye. During amplification, annealing of the probe to the target sequence creates a substrate that can be cleaved by the 5′-exonuclease activity of Taq polymerase. During the extension phase of the PCR amplification cycle, cleavage of the probe by Taq polymerase releases the reporter dye from the remainder of the probe (and hence from the quencher moiety) and a sequence-specific fluorescent signal is generated. With each cycle, additional reporter dye molecules are cleaved from their respective probes, and the fluorescence intensity is monitored at regular intervals by laser optics built into the ABI PRISM™ Sequence Detection System. In each assay, a series of parallel reactions containing serial dilutions of mRNA from untreated control samples generates a standard curve that is used to quantitate the percent inhibition after antisense oligonucleotide treatment of test samples.

[0196] Prior to quantitative PCR analysis, primer-probe sets specific to the target gene being measured are evaluated for their ability to be “multiplexed” with a GAPDH amplification reaction. In multiplexing, both the target gene and the internal standard gene GAPDH are amplified concurrently in a single sample. In this analysis, mRNA isolated from untreated cells is serially diluted. Each dilution is amplified in the presence of primer-probe sets specific for GAPDH only, target gene only (“single-plexing”), or both (multiplexing). Following PCR amplification, standard curves of GAPDH and target mRNA signal as a function of dilution are generated from both the single-plexed and multiplexed samples. If both the slope and correlation coefficient of the GAPDH and target signals generated from the multiplexed samples fall within 10% of their corresponding values generated from the single-plexed samples, the primer-probe set specific for that target is deemed multiplexable. Other methods of PCR are also known in the art.

[0197] PCR reagents were obtained from Invitrogen Corporation, (Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of DATP, dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well plates containing 30 μL total RNA solution (20-200 ng). The RT reaction was carried out by incubation for 30 minutes at 48° C. Following a 10 minute incubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of a two-step PCR protocol were carried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for 1.5 minutes (annealing/extension).

[0198] Gene target quantities obtained by real time RT-PCR are normalized using either the expression level of GAPDH, a gene whose expression is constant, or by quantifying total RNA using RiboGreen™ (Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real time RT-PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RiboGreen™ RNA quantification reagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA quantification by RiboGreen™ are taught in Jones, L. J., et al, (Analytical Biochemistry, 1998, 265, 368-374).

[0199] In this assay, 170 μL of RiboGreen™ working reagent (RiboGreen™ reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μL purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nm and emission at 530 nm.

[0200] Probes and primers to human DRAK2 were designed to hybridize to a human DRAK2 sequence, using published sequence information (a genomic sequence of human DRAK2 represented by residues 58695_(—)149492 of GenBank accession number NT_(—)022358.2, incorporated herein as SEQ ID NO: 4). For human DRAK2 the PCR primers were: forward primer: TCACGAGAAGCCAGGTCACA (SEQ ID NO: 5) reverse primer: CTCCGAACGTGGCAGGAT (SEQ ID NO: 6) and the PCR probe was: FAM-CCGTCGGCCCTTGTCTGGAAAAGT-TAMRA (SEQ ID NO: 7) where FAM is the fluorescent dye and TAMRA is the quencher dye. For human GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC(SEQ ID NO:8) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO:9) and the PCR probe was: 5′ JOE-CAAGCTTCCCGTTCTCAGCC-TAMRA 3′ (SEQ ID NO: 10) where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14

[0201] Northern Blot Analysis of DRAK2 mRNA Levels

[0202] Eighteen hours after antisense treatment, cell monolayers were washed twice with cold PBS and lysed in 1 mL RNAZOL™ (TEL-TEST “B” Inc., Friendswood, Tex.). Total RNA was prepared following manufacturer's recommended protocols. Twenty micrograms of total RNA was fractionated by electrophoresis through 1.2% agarose gels containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the gel to HYBOND™-N+ nylon membranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnight capillary transfer using a Northern/Southern Transfer buffer system (TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UV visualization. Membranes were fixed by UV cross-linking using a STRATALINKER™ UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then probed using QUICKHYB™ hybridization solution (Stratagene, La Jolla, Calif.) using manufacturer's recommendations for stringent conditions.

[0203] To detect human DRAK2, a human DRAK2 specific probe was prepared by PCR using the forward primer TCACGAGAAGCCAGGTCACA (SEQ ID NO: 5) and the reverse primer CTCCGAACGTGGCAGGAT (SEQ ID NO: 6). To normalize for variations in loading and transfer efficiency membranes were stripped and probed for human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.).

[0204] Hybridized membranes were visualized and quantitated using a PHOSPHORIMAGER™ and IMAGEQUANT™ Software V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels in untreated controls.

Example 15

[0205] Antisense Inhibition of Human DRAK2 Expression by Chimeric Phosphorothioate Oligonucleotides Having 2′-MOE Wings and a Deoxy Gap

[0206] In accordance with the present invention, a series of antisense compounds were designed to target different regions of the human DRAK2 RNA, using published sequences (a genomic sequence of human DRAK2 represented by residues 58695_(—)149492 of GenBank accession number NT_(—)022358.2, incorporated herein as SEQ ID NO: 4; GenBank accession number NM_(—)004226.1, incorporated herein as SEQ ID NO: 11, and GenBank accession number AW504293.1, incorporated herein as SEQ ID NO: 13). The compounds are shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target sequence to which the compound binds. All compounds in Table 1 are chimeric oligonucleotides (“gapmers”) 20 nucleotides in length, composed of a central “gap” region consisting of ten 2′-deoxynucleotides, which is flanked on both sides (5′ and 3′ directions) by five-nucleotide “wings”. The wings are composed of 2′-methoxyethyl (2′-MOE)nucleotides. The internucleoside (backbone) linkages are phosphorothioate (P═S) throughout the oligonucleotide. All cytidine residues are 5-methylcytidines. The compounds were analyzed for their effect on human DRAK2 mRNA levels by quantitative real-time PCR as described in other examples herein. Data are averages from three experiments in which T-24 cells were treated with the oligonucleotides of the present invention. The positive control for each datapoint is identified in the table by sequence ID number. If present, “N.D.” indicates “no data”. TABLE 1 Inhibition of human DRAK2 mRNA levels by chimeric phosphorothioate oligonucleotides having 2′-MOE wings and a deoxy gap TARGET CONTROL SEQ ID TARGET % SEQ ID SEQ ID ISIS # REGION NO SITE SEQUENCE INHIB NO NO 182425 5′UTR 4 1507 agtgactcctggcgacagca 67 14 2 182426 Start 4 9667 tcaaatctcctcctcgacat 49 15 2 Codon 182427 Coding 4 81482 aatcaaggtgtacaatgtta 55 16 2 182428 Coding 4 83804 tcacacgcatgccctatttt 67 17 2 182429 Coding 4 70845 gacaatcctgtcctcttctt 37 18 2 182430 Coding 4 88958 tgctatcctctgggatattc 73 19 2 182431 Coding 4 88817 agttttcaaagtcccactgc 27 20 2 182432 Coding 4 87046 cttgagaaatattgaggtat 55 21 2 182433 Coding 4 86992 tgtgagttaacaacatatat 49 22 2 182434 Coding 4 81370 caggctgaaaatttctccac 55 23 2 182435 Coding 4 88860 tcctgagtttgagaggaact 76 24 2 182436 3′UTR 4 89214 gatatgaaatcataacatgc 44 25 2 182437 Coding 4 81400 agaaaccatttcagccaact 55 26 2 182438 Coding 4 88846 ggaactggaagtttcttcag 74 27 2 182439 Coding 4 88851 tgagaggaactggaagtttc 54 28 2 182440 Coding 4 81411 acatcattttcagaaaccat 32 29 2 182441 Coding 4 83740 atatgctgctcagtaatata 48 30 2 182442 Coding 11 380 cacagcaaattttcctctcc 37 31 2 182443 Start 4 9649 atgttaggtgattcccaggt 72 32 2 Codon 182444 5′UTR 4 1513 tcgtgaagtgactcctggcg 51 33 2 182445 Coding 4 87085 aaactgatgaaaaagtttct 25 34 2 182446 3′UTR 4 89087 ggagtggatataaaatttca 28 35 2 182447 3′UTR 4 89218 tcaggatatgaaatcataac 52 36 2 182448 3′UTR 4 89211 atgaaatcataacatgctag 30 37 2 182449 Coding 4 88839 gaagtttcttcagggtgaaa 39 38 2 182450 Coding 11 860 gatttctggagctaaatattt 39 2 182451 Coding 4 88975 tcttttggaaaccatgctgc 56 40 2 182452 Coding 4 88885 gtcttcagaggaccttacag 25 41 2 182453 Coding 4 81472 tacaatgttattctgatgta 24 42 2 182454 Coding 4 81394 catttcagccaactcaggta 28 43 2 182455 Coding 4 9688 aggcctgaaatacttcggca 76 44 2 182456 5′UTR 4 1544 agacaagggccgacggttgt 66 45 2 182457 5′UTR 4 1609 tctagctccagccgggcgag 62 46 2 182458 3′UTR 4 89197 tgctagcaactagtaattta 64 47 2 182459 Coding 11 903 tattccacatatctgttgct 49 48 2 182460 Start 4 9660 tcctcctcgacatgttaggt 71 49 2 Codon 182461 Coding 4 81457 atgtagataataaactcctt 31 50 2 224150 5′UTR 4 1534 cgacggttgtgacctggctt 97 51 2 224151 5′UTR 4 1575 cgaacgtggcaggatccact 93 52 2 224152 5′UTR 4 1620 aggacgagttctctagctcc 53 2 224153 5′UTR 4 9633 aggtctgcttctttagtcac 11 54 2 224154 Coding 4 70814 aaattttgcagcatattctt 72 55 2 224155 Coding 11 478 tgtaaaatttctgcccgaca 83 56 2 224156 Coding 11 587 acctgcagcatattccaata 47 57 2 224157 Coding 4 81383 actcaggtaaacacaggctg 70 58 2 224158 Coding 4 81438 tcaagtatttgtttaatgag 51 59 2 224159 Coding 11 732 tattctgtggctttaaatca 45 60 2 224160 Coding 4 83830 ctggtgttcccatgatttcc 68 61 2 224161 Coding 4 87014 tcttctcccacaaatggtga 59 62 2 224162 Coding 4 87103 agtctgtggccagctgtgaa 74 63 2 224163 Coding 4 88807 gtcccactgctgtagccaag 73 64 2 224164 Coding 4 88914 gttccattacaggaggattt 55 65 2 224165 Coding 4 88926 ctatcaccacaggttccatt 61 66 2 224166 Stop 4 89040 aagtgctaacagagcaaatc 56 67 2 Codon 224167 3′UTR 4 89150 catggaaaagtgcatttaca 73 68 2 224168 3′UTR 4 89161 ctaaattattccatggaaaa 17 69 2 224169 3′UTR 11 1608 ctttcacatgtacaatttta 13 70 2 224170 Intron 4 9494 ttaaagagcacattctatcc 54 71 2 224171 Intron: 4 9613 ttatttttacctatgcaaaa 6 72 2 exon junction 224172 Intron 4 35967 agttagcataacctcacatt 10 73 2 224173 Intron 4 57224 agtgttgctctgtcgcccag 76 74 2 224174 Intron 4 58877 ctactagttatgcttgggca 0 75 2 224175 Exon: 4 81497 gttacgttacctttaaatca 40 76 2 intron junction 224176 Intron: 4 83722 tattctgtggctaaacaaag 53 77 2 exon junction 224177 Intron: 4 88762 tggtcttttcctttgaaaga 28 78 2 exon junction 224178 Exon 4 1470 agcttgcagtcgcggtttga 53 79 2 224179 5′UTR 11 208 ttatttttaccgctccggcc 73 80 2 224180 3′UTR 13 104 taaaactcaagccacttttt 62 81 2 224181 3′UTR 13 150 attaaattccaatgtcttca 75 82 2 224182 3′UTR 4 89465 ggcatggtataagagtcacc 74 83 2 224183 3′UTR 13 287 aaagacattttcaactgtaa 34 84 2

[0207] As shown in Table 1, SEQ ID NOs 14, 15, 16, 17, 19, 21, 22, 23, 24, 25, 26, 27, 28, 30, 32, 33, 36, 40, 44, 45, 46, 47, 48, 49, 51, 52, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 71, 74, 76, 77, 79, 80, 81, 82 and 83 demonstrated at least 40% inhibition of human DRAK2 expression in this assay and are therefore preferred. More preferred are SEQ ID NOs: 24, 27 and 44. The target regions to which these preferred sequences are complementary are herein referred to as “preferred target segments” and are therefore preferred for targeting by compounds of the present invention. These preferred target segments are shown in Table 2. The sequences represent the reverse complement of the preferred antisense compounds shown in Table 1. “Target site” indicates the first (5′-most) nucleotide number on the particular target nucleic acid to which the oligonucleotide binds. Also shown in Table 2 is the species in which each of the preferred target segments was found. TABLE 2 Sequence and position of preferred target segments identified in DRAK2. TARGET SEQ ID TARGET REV COMP SEQ ID SITEID NO SITE SEQUENCE OF SEQ ID ACTIVE IN NO 97703 4 1507 tgctgtcgccaggagtcact 14 H. sapiens 85 97704 4 9667 atgtcgaggaggagatttga 15 H. sapiens 86 97705 4 81482 taacattgtacaccttgatt 16 H. sapiens 87 97706 4 83804 aaaatagggcatgcgtgtga 17 H. sapiens 88 97708 4 88958 gaatatcccagaggatagca 19 H. sapiens 89 97710 4 87046 atacctcaatatttctcaag 21 H. sapiens 90 97711 4 86992 atatatgttgttaactcaca 22 H. sapiens 91 97712 4 81370 gtggagaaattttcagcctg 23 H. sapiens 92 97713 4 88860 agttcctctcaaactcagga 24 H. sapiens 93 97714 4 89214 gcatgttatgatttcatatc 25 H. sapiens 94 97715 4 81400 agttggctgaaatggtttct 26 H. sapiens 95 97716 4 88846 ctgaagaaacttccagttcc 27 H. sapiens 96 97717 4 88851 gaaacttccagttcctctca 28 H. sapiens 97 97719 4 83740 tatattactgagcagcatat 30 H. sapiens 98 97721 4 9649 acctgggaatcacctaacat 32 H. sapiens 99 97722 4 1513 cgccaggagtcacttcacga 33 H. sapiens 100 97725 4 89218 gttatgatttcatatcctga 36 H. sapiens 101 97729 4 88975 gcagcatggtttccaaaaga 40 H. sapiens 102 97733 4 9688 tgccgaagtatttcaggcct 44 H. sapiens 103 97734 4 1544 acaaccgtcggcccttgtct 45 H. sapiens 104 97735 4 1609 ctcgcccggctggagctaga 46 H. sapiens 105 97736 4 89197 taaattactagttgctagca 47 H. sapiens 106 97737 11 903 agcaacagatatgtggaata 48 H. sapiens 107 97738 4 9660 acctaacatgtcgaggagga 49 H. sapiens 108 140804 4 1534 aagccaggtcacaaccgtcg 51 H. sapiens 109 140805 4 1575 agtggatcctgccacgttcg 52 H. sapiens 110 140808 4 70814 aagaatatgctgcaaaattt 55 H. sapiens 111 140809 11 478 tgtcgggcagaaattttaca 56 H. sapiens 112 140810 11 587 tattggaatatgctgcaggt 57 H. sapiens 113 140811 4 81383 cagcctgtgtttacctgagt 58 H. sapiens 114 140812 4 81438 ctcattaaacaaatacttga 59 H. sapiens 115 140813 11 732 tgatttaaagccacagaata 60 H. sapiens 116 140814 4 83830 ggaaatcatgggaacaccag 61 H. sapiens 117 140815 4 87014 tcaccatttgtgggagaaga 62 H. sapiens 118 140816 4 87103 ttcacagctggccacagact 63 H. sapiens 119 140817 4 88807 cttggctacagcagtgqgac 64 H. sapiens 120 140818 4 88914 aaatcctcctgtaatggaac 65 H. sapiens 121 140819 4 88926 aatggaacctgtggtgatag 66 H. sapiens 122 140820 4 89040 gatttgctctgttagcactt 67 H. sapiens 123 140821 4 89150 tgtaaatgcacttttccatg 68 H. sapiens 124 140824 4 9494 ggatagaatgtgctctttaa 71 H. sapiens 125 140827 4 57224 ctgggcgacagagcaacact 74 H. sapiens 126 140829 4 81497 tgatttaaaggtaacgtaac 76 H. sapiens 127 140830 4 83722 ctttgtttagccacagaata 77 H. sapiens 128 140832 4 1470 tcaaaccgcgactgcaagct 79 H. sapiens 129 140833 11 208 ggccggagcggtaaaaataa 80 H. sapiens 130 140834 13 104 aaaaagtggcttgagtttta 81 H. sapiens 131 140835 13 150 tgaagacattggaatttaat 82 H. sapiens 132 140836 4 89465 ggtgactcttataccatgcc 83 H. sapiens 133

[0208] As these “preferred target segments” have been found by experimentation to be open to, and accessible for, hybridization with the antisense compounds of the present invention, one of skill in the art will recognize or be able to ascertain, using no more than routine experimentation, further embodiments of the invention that encompass other compounds that specifically hybridize to these preferred target segments and consequently inhibit the expression of DRAK2.

[0209] According to the present invention, antisense compounds include antisense oligomeric compounds, antisense oligonucleotides, ribozymes, external guide sequence (EGS) oligonucleotides, alternate splicers, primers, probes, and other short oligomeric compounds which hybridize to at least a portion of the target nucleic acid.

Example 16

[0210] Western Blot Analysis of DRAK2 Protein Levels

[0211] Western blot analysis (immunoblot analysis) is carried out using standard methods. Cells are harvested 16-20 h after oligonucleotide treatment, washed once with PBS, suspended in Laemmli buffer (100 ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and transferred to membrane for western blotting. Appropriate primary antibody directed to DRAK2 is used, with a radiolabeled or fluorescently labeled secondary antibody directed against the primary antibody species. Bands are visualized using a PHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 133 <210> SEQ ID NO 1 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 1 tccgtcatcg ctcctcaggg 20 <210> SEQ ID NO 2 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 2 gtgcgcgcga gcccgaaatc 20 <210> SEQ ID NO 3 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 3 atgcattctg cccccaagga 20 <210> SEQ ID NO 4 <211> LENGTH: 90798 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: 1349, 1350, 13454-13553, 28775-28874, 46851-46950, 52633-52732, 67991-68090, 85646-85745, 87254-87353, 89363-89462, 90660-90759 <223> OTHER INFORMATION: n = A, T, C or G <400> SEQUENCE: 4 aattgatgaa atgttgaaat taaggaggtc caaagttaaa cacacaaccc caaaaccacc 60 ataatgatgt aatcctgtga cccccaagag gttactagtt ttggtactgg tgctgagacc 120 tctggaatgc aacacctatg ctcatgaaag aaaagatggg aacagtctca ttgcttacgg 180 attcttatac cccagaaaag ttaattccag tgtgtggcat tttattttca tcctaacttt 240 aggcttctct agtcagccca attgttagca acaagaaatt agatagatgg aagcaatatt 300 ctccatttga caaggtttag gggcaagaca ttattaacct gggaggagga gttaacctaa 360 gtaacaacaa acagacttca taaaagggct gtgatgcatt ctataccttt atcttgggga 420 aaatgcagta ggaaagggaa acagctagct tcacaggaat ttgcttggct tactgggata 480 gaacaagaaa acctgtcctt tatatgctaa ctgaacatta agtcttttga aagcaagagg 540 acgactccac tcccagtatc ctttcccacg gaagggtttt ctgcaattat cgtaaatgtc 600 ctctgaaagc aatcctagat tacataacta atcccatata tgtatgagta tgtgtgtatc 660 ctatacatat aaactaggat tatatatcta tgtaaaacat ttgtgtcttg cacagaactt 720 tgaaaatttc cactgtcttc aaaggataac agatcttgag taggttgctg agggctttca 780 tctttttcct tagcactata aatgcaaaat cggctattca gcctattggg accctcccta 840 cactgccttc agtcagtgcc tcagaaggga caccattcgc gacagcggac gttagcagtg 900 gggtctaggt tccataaatc cccaactgat gttgacactc cgaggaacct ctgccaagtt 960 gtagcaaact gggttctcca cttacagggt acgttccggt cctaaggaag actctcaaga 1020 agagtcagaa aataggtctt tggtaggcac tgaagtttgg taactagtcg caggcagctg 1080 ggacgaattt tgcggcttcc ttctccccgg tctccggata ttcacacctc tcgaactgac 1140 ctctctgaag gtgggaagga gtacacagag cagtagttcc agccccgcgc gccccctctg 1200 cagcgccgcc ccctccgccc ctccctcgcg gcagcccgag tgcgccccac ctgcgccgcc 1260 acgccgccgc gcctccccgc cccgcctcgg gccagccagg cgccgcaccc tctcaccggc 1320 aggccccgcc ccgcacacgc gcccccggnn cggtgccccg cccctcccgt cacgtgacag 1380 acccaccctc cgcacccgtt ccgattggct ctcccgacct agggggaggg atctggcgaa 1440 cggcgatgcc ccagacgcgg ctgcagtttt caaaccgcga ctgcaagctt cggtagtcct 1500 ctccgctgct gtcgccagga gtcacttcac gagaagccag gtcacaaccg tcggcccttg 1560 tctggaaaag taaaagtgga tcctgccacg ttcggagctc cctggcgcct cgcccggctg 1620 gagctagaga actcgtcctg tggcggcccc cggcgtgggg cgggacagcg gccccctgga 1680 gggggcagtc ccgggagaac ctgcggcggc cggagcggtt agtgtcctgc gggattggcg 1740 ggttggctcg gggctgcgat ggaaccagcc gcctcggtac agagacctca tacgtgagga 1800 aggaggaccc gaggagggtt taggcggtac ggtccggctg agaggggctt ggactccgcc 1860 gcccctgagg tggctgattg ctgctggcgc ctctccgctt tgcggctgga gtccgggttc 1920 cctggggctc cgcgcggggg gcggccggcg gtcctcccac caccctcctc agcccggggc 1980 gcgtgtctgt gtctgtgcgt gtccgtgtgt ccccgtgcgc cctccacccg gccggccccg 2040 gctctcccca ggtcgcgcca ggcagggccc ccttcgcggc gggcccgcgc gggtcccttc 2100 cacagcaagg ccacgcggtc cccctagcct ctcccgagtc ccagctgcgg cgtgggcgcg 2160 ttcccatcgg gactcgtgga cgcgtcctgc tcccagctgc ctttcgtcta gagaaagttc 2220 gtgttcatct tgtgggacgt tttcagttac tgcttgggaa cagtgtttta aaaccagcga 2280 gagatcaaga cgggctacag ctgtttccgt gattttcagc gatctgattt ttgctttgat 2340 gccttgtgac ccacttagtg tgcacgactc atcctcaaac tataccacta ctggatgcca 2400 acgatttttg acatttaccc aggctctttg ttttattgta gggaaaagcg tttcatttga 2460 atttcctccg agggagaagt agagacaaag ttgaaagagg ctttatagca gctggtagct 2520 ggcattagtt tctgtctgga ctagaggcac tctgacatca atttggaaat tggaattaag 2580 aaaatacgtt tttaaaatcg taatacttat cagatttcac taatatttaa acacatgagg 2640 actgtgtatc acattcaccg attgttttgt cgacgtaatg tttacatctg tggtgctaat 2700 gataagcaga accttgccag ggacgtttga cgtggtgtgg ccactttacg ttttcaagtc 2760 tatgagaatg tctgcgcgga gacagcatag ctctgtagaa atgagtggca gcgtatgtaa 2820 cctggcattt tgaacccagg agcacaattt tattaaagga aaataaacct actttctcat 2880 tgataacact gttttttagt tttatggtga actgttcgga agtaattttc aacaagtgct 2940 tattttataa atattagacc gtgtacccct aggattgtgt attttttaag aaaactggtc 3000 catagaagcg gtgcaaaagt tttaaactca tctgcctcgg atcctcctcc tctgagcaga 3060 tgctcaatta aactttttct agtatcttaa taattggagg tattaataga tgttttattt 3120 ttgagataca tattgtacat tttagatctt tttttttttc taaagtaggg atccaaaatt 3180 gaggtgaaat atatttgctt acatggcaag actttttaaa agtagaattt ctgtaattga 3240 agaccatcct tttttgtgtg tgaatagaat ggttgcggtt tctcttggga tcattgatta 3300 gtgaattacg atttggttaa gatagaatgc gtttttagga agttggaggt ttgactaatc 3360 gctgtgttag catatgagta acaaatttga agaagataca agcattttta tggctgacgt 3420 ttctaatcag ataattttat ttttaagctt gctctgtttt acttttgtta agtgaacatt 3480 ttaacatgtt ttaaagctct ttgataatta taagggagaa tttccttatg aaaatatttt 3540 tgctttaatt ttagagacac aagaagtcaa ggaattcagt taataacact ttcacttaat 3600 cgttaaattg ccttaaaatt gcatgcatag tatattgtaa ttcacatttt ctgtatgtaa 3660 gattggcagt taagaatagt attctggccg ggcgcagtgg ctcaagcctg taatcccagc 3720 actttgggag gccgaggcgg gcggatcata aggtcaggcg ttcgagacca atatggtgaa 3780 accccgtgtc tactaaaaat acaaaaatta gccgggcatt gtggcgcgcg cctgtagtcc 3840 cagctactcg ggaggctgag gcagaagagt tgcttgaccc ggaaggccga ggttgcagtg 3900 agccgagatc acgccactgc actccagcct gggcaacaga gggagactcc gtctcaaaaa 3960 aaaaaaaaaa aaaaaaaaaa atagtattcc taggcttact acttttaaag gatactgatt 4020 gattcttctt ttcataaaaa aatcttctgc actgtatggg tatttgtttt gtcagaaaca 4080 ttagcagtca tttatctact acctttccac ttaggaaaac ttcaaatgtc agttgcttta 4140 gtttttcata atattgatga caaaattcag tgattttatg tttaagtact gtagtttgta 4200 ttctatgctg ggctgttatc aaaggctatt tctttgaata tttcagttaa gaaatgttct 4260 tataaagtta caagcatgta tttttattta aatatgttgt aatgttgcaa ttttcagggc 4320 atactttatt aagtttttta ttatcctttt tccagaacat ttaacctcaa acttcagtgc 4380 catttaaaac atatatatta gtttctttta atatttatat gcattgtatt ttggttcatc 4440 cctgaccctt tgcaattact ccaaaagagg gtgtagtgat actgtaagca ttgaaaaatt 4500 ttaacagtga gatggtaatt tcctctttaa tccttaaagg cagattccat tttgcattgt 4560 aatagttact gaataaatag tacttaagcg gtgctattaa gtaaacaata ttttacaaca 4620 gaaacccaat tttccgtctt gtgagtgaca agcaccatgc atttgttgcc caccccggaa 4680 tcccccactc tatttccgtt tttcaacctt ttatcctggt tcacaattaa caggtttaac 4740 tctttttctg gaggtgggaa gacacagcat taggccttag ggtcttggca cagagtcagg 4800 cctaggagtc attgcctgac acttcagccc ttggatagta tatactagaa tggttggatt 4860 agagttagtt tagctgactt cctattgtga tttttagagg attaagttta tgaattatat 4920 tttctgttca aaatatttta aaaaacaaaa cctgttggca gaatacatat gaggaatata 4980 atctcttcct tgaaattgaa aatcatcatc atttccatct tgataccaat acttctaatc 5040 aatgactaat taaaaacaca tacttctgcc acaccaggag aaagttcttc ctttactttt 5100 gatggacgtt aaatctcttt ttcagtttcc tagggaaagc ttgctagcag taaatgcctt 5160 aaatgcctat gtagaaagga gttgcttccc ttagaatctt cgggcttact ttgcttcctg 5220 ttgcctttcc aatgtacttc atgatgatct agacttatca gtgataaatc ttgataggac 5280 acttacctga atctcctgat ttttggaggc tcttaaaaga gtgatgttct taactgcagt 5340 aggtatttgt attagtagct tatattaaaa acaagtagcc accttgtctg gaaacagtgg 5400 taaaaacaaa aggtctattg gcattacatt atagtatatg acaaaataat tttatctgtg 5460 tttccagact ttgtggctac ctgtttagaa ctgacatttt gtaagtgcaa tttaaaatag 5520 ttactttatt tgtccaaatg ctgaattaag cagagactag ctgcattcag agctccagta 5580 ctgtaaattc aagatggatc tcaagttctc ctaacccctc gaagacatga gatataccta 5640 caaattcttt tttctatatc tcataattct tacaattcag ttcatcaaac atttttaaaa 5700 ttcctaccgg gtggcagata ccatgctggt tgcttaggca caaaagtaaa aggatgctgc 5760 cctttaaaca ctgaacagaa ttgcaatcta acatgttaca ttttgaggta aagaagtatg 5820 ccaaggatcc tcttatttga agcataaagg agaagccttt aaatcagcct gtggttttta 5880 ggcaaagctt cctgggggag aggccattct gtgttgagtt ttgaaggatg tgtaagaaga 5940 gttagccatt taaggaaagt agaaaggctt tttcaggcaa agaagtataa caaagatgtg 6000 gtggcaaaat atgaaatatg agtgcggcag gaaatgtagc tggtgtatac ttggcctcag 6060 aggcctttat gtgtcatgct gtaagtactt gaaagcttgt agatactggg gagctaatga 6120 aggaatcaaa acagacaatt tgattttcta aatgtcttta ttattgtata ttctattttt 6180 taaaaacttt cttatttgat aattttgaaa taaatatcat gataaatact ttttaaaata 6240 gagctagagg tttatagaga tagtcttgct tttgttcaca tcacacttgc tttctgcatt 6300 caagtaagtg aaggcccaga tcaagcctaa catttccttc ttttccttcc ttcactgatc 6360 tacttccaaa ttcttttggt aattattata atgagaatta atgaaaaaaa tctttaaaaa 6420 taagtgagat accattattt gtagtagtac tgtgtgatta aaagcttaat tgtgtcttac 6480 cttctattac actttttata ggatctaatc gtgtatacac atgcatataa atttacttac 6540 caggtccctt aagatttttt ttaaatgcta taacctgata aaatgttatg aaatgaattt 6600 caaatggagt gagacatata ggtaatttag aatcggtaat atgaagtgtg gtagaagaca 6660 agaaaaggcc tgggaattgt tggagttaaa gagaatttgg tgggaaattt aggaagatac 6720 aagaaggagc gacagagagc tgactcataa cctgcatgta ccttatatga agggcaacca 6780 gatgtttgag gagggcttgg tggacaaagg tgacattcat gctccactat atgcaaaact 6840 ggccaaagga gaggaaatgg aaaaatatct tggctctgac aattggttca gatgcagtaa 6900 ataaaaggaa aatgaactta agaaatgctt aatggatgcc taaagcagct tgtgatgtag 6960 gtatattttt aataaataga tgaacttatg aggaaattgg tacccatatt gatctttcag 7020 aacaatggaa aaaaatcagt gaaactgagc gctatcagaa ctaagaagat acataagtag 7080 ttaaaacaga aaacaaaaat acttttaaga atgaagttta gactaagttg ggatgagaat 7140 tttgtacatg gaaacagtac agctgttcaa caatggcagg gaactgcagg gagagtattt 7200 ttgggtagaa tgttaatttt gttttgtgga acaatagcga tatgcccttg tgacttgcac 7260 tgtacctgtg tcagtggaca tgagttcagc atattttaat ttttaatcct tgcagcactg 7320 gaagatcaat tcttttatca tccatacttt agaggcagca aaatcgaagc tttcttcacc 7380 agtaaaaggg aatgcgaagt aaatcacaac aaaattccat ttcaccttca tcacactaga 7440 atagccaaac ttaaaagtct gaattgggca agtattgtca aagtagtgat aaaccgtccc 7500 atccagtgct agtgggagtg taaactgggg gtggaaagaa ggggctatgt ctaatatgtt 7560 ttatttttaa tttaaatgaa atatgaccaa gtaataagat tggaaagtaa atgtttgtat 7620 atagtgttcc ttatcttaca tttgaaatat tatatatttt gttttctttt taaataactg 7680 tgttgccgca gatcaggcga cttaacagaa gtatattttc tcaacgattc tggaggctag 7740 aaatctggga tcagggtgtc tgcggggttg gtttcttctg aggcctgccc tcttggcttg 7800 tagatggctg ccttctccta gtgtgttcac atagtctttc ctctgtgtgt gtctgtgtcc 7860 taattggccc actttaatga cctcatttta acttaatcac ctttttaaag gccctgtctc 7920 caaagacagt cacattatga gatacagggg ttagggtttt tcaacttagg aatttggggg 7980 aggacacaat tcagcccata acaatagcaa agttaaataa tttaattaag gtctcatagc 8040 tggtatgatg gatgaatgga attcagaccc aggtgtcttt cagtctaaag gttattctca 8100 gaatgctaca ctacaaccct ttgactccta gagatgtacc ccaaaactag ttagagctgt 8160 ttccccaaag actaagagat gtagagaaaa caattttact atctagttat gtgaaagcta 8220 taattttaat ttaatttttt gagacaaggt ctccatctat cgcccaggct gaactgcagt 8280 gacgcgttca taatttgaga ataacaccaa gaagagaaaa tgtagaaact gaaaacgctg 8340 aactctaatg acactgcttt ttggctaagt agactatagt tggcatttga atttaagaag 8400 ataaagaaca gtacatataa gtagaaaata ggaaaaaaag atgtcagagg aggaacaaat 8460 gcttagcggg ttactacagg agggtgctaa aagaataata attgatattt tattaagtag 8520 gcatagcaat aagtcataag tgtcttgaaa tatagtatct gttattgttt tatgaatgca 8580 attaatataa tagttttgat aacactgaat gcatataaac atcacaaatt tccagataca 8640 tatatgagct tcagatacag aacttcgaaa taaagcttta tgtataggtt agtcactctt 8700 ccaacaaaat tattgagcac ttcatacata gcaggctctg tgctaggtcc tgtcaaagga 8760 gggactctta agacctttga gagtttctga gtttagtact gaaacatagc caatccgtgg 8820 aagaagttct atttcttgta gtccttcacg tcagtaatat aactcacaat atgtgcattt 8880 accaccaaat ttctttagcc atgtgaaaat aagtttaaaa tgaataatta ttatcttgtc 8940 gtattaaatt acctattatg tgaaacctat aactttattt atttatttat ttatttattt 9000 atttatttat tgagataaga tctcgcttta ttgcccaaac tgaactgcag tagcacattc 9060 atagctcact gcaacctcaa acttctgggc tcaagtgatg ctcctgcctc agcctcccaa 9120 gcagctggga ctacaggtgc gagccactac acccagttaa tttttaaatt ttttcataga 9180 gatgggatct cgctatgttg ccaggactgg ttttgaactt ctggcctcaa gtgatcctcc 9240 tgcctcagcc tcccaaagtg ctgggattat aggcgtatac caccatgtcc agctgaaacc 9300 tctataactt taaagaatgg agttttatga tgaaactaag tgattgtttg cattataatt 9360 ttatcataac tatttttttt aaattgagag gatttttacc gtagttttat tctgaaatga 9420 agtcagtaac ttcgttcaag tgccatagtg ttattatggt ttatgtgtat gctttttgtt 9480 ttttcctcct tgtggataga atgtgctctt taaatctttt ttttttttag ttgtatttta 9540 tagctatatt tggagctgtg taatgtaatg cacatcctgc ctgagataac atcttttctc 9600 tgtattctct ttttttgcat aggtaaaaat aagtgactaa agaagcagac ctgggaatca 9660 cctaacatgt cgaggaggag atttgattgc cgaagtattt caggcctact aactacaact 9720 cctcaaattc caataaaaat ggaaaacttt aataatttct atatacttac atctaaagag 9780 ctagggaggt aagaaaacca tatgacgtgt atgccatcta aattcaaatt ttggatttgt 9840 gtttttataa ttctataaaa ctcacttggg atcattattt aacaatgatt aggaaggaaa 9900 gttgtgatct ctgttgctgt tacctagggt ccagtcttaa ctgacttgaa aatcaaatct 9960 gtggagggtt tagtgaactt aaatcataca tcctacctag cctcagagcc tctgatagga 10020 ggtctctgtc gttacatgag gctgtgcatg ggaagaggca gctcagcata gtggactgcc 10080 gagttcaagt ttggactcca ccagttacta ggtgtgtgac ttcaactaaa taactgtacc 10140 tcttttttta atttttgttt gtggcggggt ctcactctgt tgctcaagct ggagtgcagt 10200 gacatgatct tggctcactg caacctcctg tctcctgggc tcaagggatc tgccacctca 10260 gcctcctaag tagctgggac tacaggcaca caccaccaca ctgggctggt tttttgtaga 10320 gacagggttt ctccatgttg cccaggctgg tctccaactc ctgggttcaa gcaatcctct 10380 ggccgtggcc tcccaaagtg ctgagataat tggtgtgagc ccctgcgccc tgcctactgt 10440 acctcttagt gctgtcgttt cctcatctga aaagtagggg tgaagataat tgtgtctgtc 10500 ttatatggtt gttatgagga ttaagttagt taatatttgt aaagtgccta gtacagtgct 10560 ctgtaagttt ttgttaaatg aaatattttt ttaaattcct agggtattgt gcttggctta 10620 aaaaaaaaaa aaaaagaaga agaagaagaa atagggagat tctccccctc tcctaagttg 10680 aacaaaaaga atgaattcat atttcatagt ctgattctat gtcacaacaa atgagtggtt 10740 tataagtaaa aagcctttac aaattgtttt ctcttaacca gtgtataaac tctgtacggc 10800 caaggagtga catgatatta tgaaaccatt ctgtactacc attttaatat atgaaaagtt 10860 ataaaagata taccaagtta tttaagttat atattcagca tcttttgagc tttaaattaa 10920 aggtgcttaa tgaagggttg gaagatggta gtcatcatgt attttcagtt tgattttagt 10980 tctggaggta aatgttttgt tgtatgttaa atggtttact aagtccatat ttgtgttgat 11040 aaatgtgcag caaaacaatg tgattgattt ttgtctttga aaatctcgat gtgagaagaa 11100 aagttgactg aatcatgaaa agtaatttca ttcttggtgg ccttttacct taagggcaga 11160 ggcaagtcct ggctttttca cgttgcagat gttggatgca gcctttcttt tgcaaaataa 11220 gaattataat atctggccag gtgtggtgac tcacgcctgt aatcccagca ttttgggagg 11280 ccgaggaggg tgggtcacga ggtcaggagt tcgagaccac ccttgccaac atagtgaaac 11340 cctgtgtcta ctaaaaatac aaaattagcc aggcatggtg gcgtgtgcct gtagtcccag 11400 ctactcagga ggctgaggca gaagaatcac ttgaacccgg caggtggagg ttgcagtgaa 11460 ccgatatcat gccactgcat tccagcttgg gcaacacagt gagactttgt ttcaaaaaaa 11520 aaaaaaaaaa aaaaaaaaga attataatat ctacctcact gaccaagaat taaatacaaa 11580 agcacatttc ataattcctg gcatactaat ttaatgtgca aaatatttgg gtgttttaat 11640 ggtgtattaa aagatagagt ttttagaacc ttttaaaatg tttttaagaa attgttctaa 11700 atcttaatta cttcattttg cattataata ggaagaaagg tttttttttt ctttttttct 11760 ttttcttttt tttaagtttc ctgtatctca cattgaacca tagaatctct gcattggaaa 11820 gacttcagtg gtccgtggtc ttactaatac aatgtttgta tgcagcgcag caatctttcc 11880 tataatttcc cgaacaaact gttatccagc ctcagtttga atgcttcagc gatggggaaa 11940 tgaatattat gcaagtcttg aacagtacac acaataaaag tcactttctt ttctattttt 12000 ctgagttact atttgatgag taaatttact tacatttttt aaattaactt ttcaaaattg 12060 gggacattat aggagctaaa aaatctataa agtatttttg ctttgagggc tttggataat 12120 ttaaactttg aggatgatat tagtaattct aaattgcttt ataaatgagg tgccaccata 12180 aataaagttg aattaggcat actttttaga attttagatc aaaaactcat gatagaattt 12240 gattaaagca aatttagatt gattgctaaa ttcccatttt aaaatatctt tatgaaagag 12300 accaagtaac atctttcttt tgaattgata tgggctaatg agtcacatgg taaatattga 12360 tatgtttaat tattagatct tgataaatta gaaatcatgg cttttcatct gccaagaatg 12420 tactgaaagt tctttcaata ttgttaccaa ttttaatatc ctatgctttt gtgaaatgga 12480 aattagaatc ataatcattt cttctgaggt ttgcacattt tgaaagttat ctttctgaat 12540 caactttttt atttcagagt gttagtgttt aataattcat tttcctttag caaattaaag 12600 gaaattatgt aaaagttaac agttcatttt ttactattct caaaaaactt gaattgttcc 12660 tgttttttaa catgttgggt aagttttttc tacccctccc ccaactgagg caaaggtttc 12720 ataggacttc ttgtggttag aagggtgagg cagattcaga aataaacgtc agcttccttt 12780 gtggcttacc ctcccgcctc ctttcacatt ttattgtgta ttctagaatg cagaagggga 12840 atgcagaagg gtaactacta gaccttttcg ctgctgggaa atctagttag ccttgccttt 12900 ttagggtata acttaaggat tatttttaaa tgctttaact tttcttaaat gtttcaatta 12960 catttggaat agaaacatat tttaaagatc attttttata cttttaaact atagcatttt 13020 agaatggaaa acaaatattt ggtttttttc ctaagtcatc tgataattat ggtaaaaatg 13080 tttaccttca tagtatataa atgaatacac acgcacacat atatataatt ttattggaat 13140 ctcaatcttt taagaaatac aaacacttta agtacaggat acagaatagc acagtatatg 13200 gtattatagt gggtacatat ttctggcatt cattcaacag atatttcagt tcctattatg 13260 tgttaacaaa ctgtagattt tataattacc ataataatta ggacagatat ggtccctgcc 13320 ctcatggagc ttttacttag tggtgaacgt tgacaaataa ataaatcatt atccacatca 13380 tttggtcacc tttgccctgg attaatgagt actatacttg tttaagaaag ctcttgggct 13440 ggtgcagtgg ctcnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 13500 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnncttaaag 13560 ttttttattt tttgatgcat ctttgtttag ttttggtgtc agagtaatac tggcctcata 13620 gaatgagctt tatgaaagta tttcctcctt gcattagttt gttctcatgc tgatatgaag 13680 aaatacctga gactggatga cttataaagg aaagaggttt aattgactca cagttctgca 13740 aggctgggga agcctcagaa aacttacagt catggcagaa gggaaagcaa acatgtcctt 13800 cttcacatgg cagcaggaag gcaaagaatg aatgaagccc ggtggaaaag caccttataa 13860 aaccatcaga tctcctgaga actgactcag tatcaccaga acagcctgag gataactgcc 13920 tctgtaattc aattacctcc caccgggtcc ctcccatgac acatggcgat tatgggagct 13980 acaattcaag atgagatttg ggtggggaca cagccaaacc atatcgcccc tcctctaggt 14040 tttttttttt tttttttttt ttgtattagt ttgagtagga aggtttggta gaattcagca 14100 gtgaagccat caggtcgtgg gcttttcctt gttgggacac tttttattat ggatttgatc 14160 ttgttacttg atcttgttac ctgttggtct gttaaggttt tgtatttctt tgtggttcag 14220 tcttgatagt ttgtatgtgt ctaggaattt gtccatttct tctaggtttt ccaatttatt 14280 gggatatagt tgtctttagt agcctctaat gatcctctga atttctgtgg ttttgattgt 14340 catgtcttct ttttcatctc tgattttatt tatttgagtc ttctctcttt ttttcttagt 14400 ctggctaaag gtatgtcaat gttgtttagc ttttcataaa accaactttt cagttcatct 14460 tttggtattc tttcctttat ttcaatttca tttatttctg ctctgatgtt tattatttat 14520 tttcttagac taattttggg tttgatttgc acttgctttt agaattattt aaatcagagc 14580 attgcttatt tgaagttttt gtacttttta atttagccac ttgtagctat aaaattccct 14640 ctttgtgctg cttttgctgt gtcccatagg ttttggtatg ttatgtttcc attatcactt 14700 cctccaagaa attttttcaa tttccttctt aatttcttca ttgactcact gatcattcag 14760 gagcatattg tttaatttcc atgtgtttgt gtagtttcca aaattcctct tgttactgat 14820 ttcttgtttt attccattgt ggtcagagac tatacttgat attatttcaa ttttttgaaa 14880 gttttaagat ttgttttggc atctaatata tggtctattc ttgagaatga tccatgtgct 14940 gcagagaagg atgtgtattt tgcagccatt ggatgaaatg ttctgtaaat atctattagg 15000 tctatttggt ctatagtgca gattaaatct gatgattctt tgtttatttt tttgtatgaa 15060 tgatcttttt ttgtatgaca tatgacatac aatccttttt tgtatgacat gttttgtatg 15120 aatgttgaaa tgggatattt aagtctccag ttattattgt attgaggtct atctctctct 15180 ttagctctaa taatatttgc ttaatatacc tggtgctcca gtgttgggtg cctatatatc 15240 ttcaattatt atatcctctg gctgaatcag cccctatatc attacacaat gaccttcttt 15300 gtctcttaat agcttttatc ttgaaatcta ctttttctga tatacgtgta gctactccgg 15360 gtctcttttg gttttcattg gcatggaata tctttttcca tctctttatt gtcaggctat 15420 gtgtgtcttt gtaggtgaag tgtgtttctt gtaggtgaaa gatcactggg tcttgttttg 15480 ttatccattc agcaaggcta tgttttttga ttggagagtt tagtccattt acttttgatg 15540 ttattgttgt tattgataat tttgattgat aagtaaggat atactcctgc cattttgtca 15600 tttgttttct ggttgttttg cagtcttctc ttccttcttt ttctcctttc tgtcttcctt 15660 ttagtgaagg tgattttctc tggtggtatg ttttagcttc ctgcttttcg ttttctgtat 15720 gtctgttgta tgtttattga tttgaggtta ccataaattt tgcaaataat atcttataat 15780 ttattatttt aaattgttga caacactgat tgcataaaca aataagcaag aaaaaagaaa 15840 attaatttaa aaatcttatg ttttgactgt atccccctgc tttttaacat tttgttgttt 15900 ctgcttatat cttactgtac tatctgtctg gaaaagttgt agttattttt taccagttca 15960 tctttctgcc tttctactta acatatatat atagtttaca caccacaatt acagtgttat 16020 aatagtctgt gtttttctgt ttatttacca taattgatga gttttgtacc ttcagattat 16080 ttattattgt tcattattgt cctttgcttt cagattgaag aactcccttt aatattccct 16140 gtaggacagg tctggtgttg atgaaatccc tcagcttttg tttgtctggg aaagttcttc 16200 tttccccttt atgtttgaag gatattttca ccagatttgc tattctagga tacaagttct 16260 ttttccttca gcactttaaa tatgtcatgc cactctttgc tgggtggtaa ggttctcact 16320 gaaaagtctg cagtcagaca tgttggagct ccattggggg ttgtttattt tctcttgctg 16380 tttttaggat cctttcttta tttttgacct ttgggagttt gattattaaa tatgttgagg 16440 tagtctttgt tgggttaaat ctgcttggtg ttctaaaacc ttcttgtacc gaaatattga 16500 tatttttctc taggtttgga aaattttctg ttattatcct tttgaataag gttctaccta 16560 tctctctcta cctcttcttt aaggccaata actcttagat ttgccctttt gaggctattt 16620 tccagatcct ataggtgtac ttcattcttt tttatccctt tttcttttgt ctcctctgtg 16680 tatttttagg tagcctatct tcaagctcac taattctttc ttctttttga taaactctgc 16740 tattaagagt ctgatgcatt cttcagtatg tcaattacat tttcaacccc agaatttctt 16800 cttgattctt ttttcattat ttcaaattct ttgttaaatt tatatgatga gattctgaat 16860 tccttcatgg tgttatcttg aatttcaaag agtttcctga aaacagctat tttgaattgt 16920 ctgtctgaaa tctcacatat ctctatctct ttgggtttat ctctttggtg ccttatttag 16980 gttgtttgat gaggttatgt tttcttggac agtcttgatg cttgtggatg ttcattggta 17040 tctgggcctt gaagagttag gtattatacc tttagcattc tgggcttgtt tgtacccttc 17100 cttcttgaga agactttcca ggtatttgaa gggacttaaa tgttgtgatc taagtttttg 17160 gtcacttctt ctgtgtctgc attaagggat accccaagcc cagtaacact gtggatctca 17220 tagccttgta gaggtactgc cttggtggtc ttggatataa tctggaagac ttgtcttgat 17280 taccaggcag agactcttat tctcttccct taatttctcc caaataatta gagagtctct 17340 ctctctctct ctctctctct ttctgtctgt ctgcctctct gtctctctct gtctgctaag 17400 ctgcctggaa ctgggggagg ggtgacacaa acacccttgt gaccatcacc actgggactg 17460 tgcttggtca gaactgaagc cagtgcaaca ctggttctca tttaaggccc atggtaacct 17520 cttacttgct actgcctgtg tttattcaag gccctagggc tctacaattg gcagatggtg 17580 aagccatcca ggcatgtgtt cctcccttca ggtcagtgag ttcccccagc tccgagtgtc 17640 cctgagatgc tgcctgggag ccagggcctg gagtaggaaa ccttaggaat ctatgtggta 17700 ctctactcta ttgcagctga actggcatca gaccacaaga caaagtcctt cccactcttc 17760 cctccccttt tgataagcag aggagactct tatgtccacc accaccatag gtccttgggg 17820 agtattgcca ggccacagcc aatgttcact taaggcccaa gggctcttca gtcaggttgt 17880 ggtgcatgtt gctaaaccta ggacccactt tcacaaaagt gggctcccct ctggcccaag 17940 ataggtccag aaattcctcc taacagccaa ggcctggaat tggggatccc gagatcccac 18000 ttcatactct gccacactgt ggccgagctg caaggctgag gtatctaaac tgcaagacag 18060 agtcctcttt actcttccct ctgcttttct taagcagaag gagtctctct tggtagccac 18120 tacagttgtg aatgtgccag atcacacctg aagccaggac atctcagagt ctcatccaag 18180 gcccagggtg tttcctaact ggctactgct cctgattatt cagtgcccaa gggctcttta 18240 gtcagcaggt gatgaattgt gccaggactg catccctccc ttcaagccag tgggttttct 18300 tctggcccat gtgtgtctag aaatgtcttc caggagctag ggcctaagat gggggcctca 18360 ctactctacc cattgcccta tcctgctgtg gctgagctgg tatccaagtt gcaaagtcct 18420 ctttactctt ttctcttcct ctcctcaagt ggaatgaagg ggtcttcttt ggagcagtga 18480 gctgtgctgc ctggggttgg gggagggatg gcacaaccac ttctttagct aggtatatca 18540 ctaggtcatg tgcacctcaa gcccactggc tctgacccca cacggcacta aaacttgcct 18600 aggagttgca gttcttgtgg cctagactgc ctttcaagtt gtttagagcc tcagaacact 18660 ttagcccatg gtggcaaggt ttgccaaaac tcaggttgta acatctggaa tgggtgattt 18720 ccctctggct agggctgctc taagtggtcc ctccatgggc atttgctgag gtctgtgcat 18780 gttggcagca ccgagttcca acacaaagtc ccactctccc tccctgagaa cagagattct 18840 cagtgcctag cagctgatgc ggggggccag gggagggatg gcatccacaa ttcaagacta 18900 tctttcctac cctcttcggt gtctctttca gtaatatgaa gttaatacca ggtactgtga 18960 tcattcactt gatgtttggt tttcatgaag gtgcttttgg gtttagatgg ttgttaaagt 19020 tggtatttct atggggagaa tgatcagtgg aggcttcgat tcagccatct ttctctgcct 19080 cgatcctgat tttaaagtat ttaagtgcgc ccagcaaatc ataaaaatca ttcaaaactt 19140 tctatacaca ggaaattata aaaaagagtt aaagtgatta catatgtatt tttaaaaatc 19200 attcacttaa atgaaaattt gttaaaatca ttagaaccaa tttttacgta tcagtcttta 19260 aggagagttc tgtcctttgg aaagtatttg tacttggatt gaggtccttc atatgtgact 19320 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtaaaatata 19380 catacatgcc tatatatatt tttagaatat ttttatattt tatattattg ttggacatca 19440 agtgattctt tggttatgaa tgattataaa tgtagttgga gagttaaact gtaaattcct 19500 gttcattaaa ggaaaattcc tttgggaaaa tgaatattga atatggagag tatcatcatt 19560 tatttatcct cctagctatt actggaaata tttaggaatt tggggattat ttattgtgaa 19620 tttctactgt acataacagc ctttgacaga attagcaaat aggattcatt gcctgatatt 19680 tcaaatattt ctatatttta aaaaagatta ttaccacaag ggcaaaacag ttggtgaatt 19740 tattcagtat agaaatcagt cctttattat cagtaagggg aaataaaaat cccagtcatt 19800 tgactacata gatagctgat taactaaatt atcaaaaata tattttcagt tagacatttt 19860 gttacttgtg gaaaccacca tggtttgtat cctgttggtt tggatttaca acataaaagc 19920 atttagtcta attagaataa tatatcaatg aacaatttct aagcaacttt ctccttcaaa 19980 aacattgtta ggtcttcatt atttccttcc ttctcccttt ctttcttccc ttccccttcc 20040 ccttctttct tgaaatggag tctcattctg tcacccaggc tggagtgcag tgatgtgatc 20100 tcagctcagt gcaaccactg cctcctgggt tcaagcaatt cgcctgcctc agcctcctaa 20160 gtagctggat tactggcgcc cgccaccaca cccgtccaat tatttatata tttttagtag 20220 agacggggtt tcaccatgtt ttgaactctt gacctcaggt gatccgcctg cctagacctc 20280 acaaaagcct agggttacag gcatgagcca ccacacccgg ccgattttta atattttctt 20340 aaaaatacaa aggaagggtc agccgcggtg gctgacgcct gtaattccag catttgggga 20400 ggctgaggtg ggcggatcac ctgagattag gagttcgaga tcagcctgtc caacgtggtg 20460 aaaccccatc tctactcaaa ttacaaaaca aacaaaaaac aacctagcac atgcctgaat 20520 cccagctacc tgggaggctg aggcaggaga atcgcttgaa catgggaggc tgaggttgca 20580 gtcagccgag attgtgccac tgtactccag tctgggtgac agagtgagac tccatctcaa 20640 aaaaaacaaa aacaaaaaca aaacaacaaa caaaaaaaac tgtataggtc caaacagttc 20700 agggaaagag gaaaaagtgg aatttacagt aaagtgcatc catgtttagg ttatcacatt 20760 cccacgcgcc caatacccac ccataaaaca agaatttcat tccatgacac aaaggagctc 20820 ctattacctt gaatggtagt ccagaagtat tggtaccagg gggactggaa ctaatgaaga 20880 aatgttggtt gtccttgagt attcagtact gggataaatg aaacaaagcc atctgctgcc 20940 ctaccactct ggtctgctgc cctaccactc tgctgcagtg ctgctggccc ctcagcccct 21000 tacccttgct ccttcttgcc actgggccca tgttctctcc acatgcagcc ctctccactt 21060 gtttcattcc ctcgtttcct tctatttgtg cttctccaag atacgggaac taccctccac 21120 ctctcactac ttcacatcct tatgatatgc tccgagcagt gtgcctgtgt agcagttgtc 21180 tctgttgcaa gctcgaagtt ttacatttgt tctggtggtt atttgggtaa aagagactcc 21240 ccaactggac tgcaaactca gggagggcag agcccatgtc tgtctttcat cacagtgtat 21300 tgccagcacc tggcacagtg tcaggcccat agcaggtgcc cataaaatgt tactctgaaa 21360 gagtaactct tcactcagat agtatctgct tcatatctat cttctgtgaa cccacagcac 21420 tcctccctgg ttttccatgt gttgttgagc attttctttc tttttctttt tttttgttgt 21480 tgtcatttta gtgagatgtt tgggagggat gagaaggaaa ctatgctgta gtccctgatt 21540 ttgaaccaaa agtgttgact tctaataagt ctgtgcatag ttatacagtg tatttccgca 21600 ggctgtttcg tgacatgagc tgttcgttaa aatgtcacct gtaatttttg ttttccctca 21660 ggcttacatt cagaaagtgg aggtaactga tatgattgaa ctagaacaga gattagtgga 21720 ttccaaaaac tgcctcgcct tcctcatcga gtatgtcaac ttttctccag cagacatgag 21780 gctaaataat agtgttttcc agtggtatgg aaggatggga gaaatttttg aagaacacag 21840 gaaaatcatt gaagagaaaa tagaacaata tcaagaaggt ctgaaggttg gcatcttaga 21900 tattttattt tcattttctg gaaatgtttt gtttttatct ttttatgtag cattgtcaat 21960 tattttattt gagtgcatac ttttttagct ttctcatact ataatgctat aatctcacta 22020 taaatattag tgggagacag taatatattg gatcttttat aatgcttatt ggatgattaa 22080 ggagtcagta gtatattgat caatatgcag tcatttcttg atttttcaag ataatgtgaa 22140 aaaaatggaa tatatcgtgc ataggtgata gttaataaaa atagtattta tcttacataa 22200 tttatagtac tattttcggt ttttaaaact tgtttgagta tcaaagctcc cttgatgaaa 22260 tgtgctttca tacctcataa ttgctcactc tcatgagcat tgtgcccaga atcgtcactc 22320 taacattcag taactccagc ttttttcttg ggggggaggg ggcagtcttc catataagat 22380 gatttatgat ttgcccattt cactgtccag cctcattgcc tacttctccc cactcctcac 22440 tcatgacttt tcagtaaccc tgtcaacttc tagatgtctg ctctcctatg cttcttgtct 22500 cctgcacatt ctccccatca ctctgagcac acccccgccc cacatgtgct tgttcatcta 22560 gcacactcta ttcatttttc tgagaagccc tctcagcaga cctcagtgct acttgttctc 22620 tcctgtgctc tcactttgac cacatctcta tcggacaatt atctgatcat tatgttagtg 22680 tttgtaagtc tgcctcctga tgggatcatc agctccttga gagcagggct gtttcattat 22740 tacatttctc catttatatc cacttcccag cacagtgact gacacacaga aatgctgtga 22800 gcattccata gctactgtat tagtaaatga cttaatattt acagtctcat tttacaggtg 22860 gaagagtaag tttcccaaag attaagagac ttgttctagg tcagccagta cagatgtcct 22920 ttaatagcaa gccatgcttt ctttctatta cattatagca aatataattg gcaaaatatg 22980 ttgtttggag aataattcat aggcttaaaa aactcttcat tttaaactgt agtttgtatt 23040 tttgttttac attaagggta tatcagattt ttttaaaaac tgcagaaaac atcatgagta 23100 gtaaatatta gatgtttaag tttcagtaaa ataggctctt ggaaaattgc taagtggccc 23160 agaattattt atgctatgtt ttaaagtata ctattccaca aactacacac actgttgatg 23220 aacttttcct ctcattttcc ctccccgact gggctagagc aataagtttg ccaggaaact 23280 agagaaaaga ggacataaat cagcctagat aatctataaa atagaaaatc tatttttgaa 23340 taacctaaat tttttgaata ttttattgag ctatttttta gagccattta atttggaatg 23400 ccttaccaat accaattatg taataaggct tttttactta aaacatacat acctatctta 23460 tattagaaca ctatagtttt taagtgttta aatctggatt tatttacctt acgtgtttct 23520 tgtgaagaga gcccattgga caagaaaaat aaccttggaa tatcttgctt tagtattgct 23580 gattgaaatg atatcagagt acaattaaag aaattccgtt tttttcagtt tataaaaata 23640 gcataggtaa aaattgaaat tttcttttag tcccattctt gattattaag agatttttca 23700 aatgctgata aattatttgt ttgcataaat tcttcagcat attaaagatc accattgtct 23760 aacattttta ttaattttat tagctagaac ttcttacata gataatacag agttgacttt 23820 ttctaatctg tgagttagag gaatctaaat ccattaattt aaagttttac aattctgcat 23880 attttagatc tcgttcattt tccagatcat ccttttatgt tttaccagtg acaaaatata 23940 ataataattc tgataactcc tacaatgtta aaattttttg acagcaaggg gttttaactt 24000 ttaagaatat taacaacaaa ttgtttttat tagttacggt gtgaacggtt tgtggaggaa 24060 ttggagagtt atgctaagca atcagaagaa ttttattcat ttggagatct tcaggatgtt 24120 cagcggtacc taaaaaaggc tcaaatactg aatggaaagt tggatttagc tgcagataag 24180 gtataattca aaaattctta aaaagatgaa ttagttcaat tgaaaaagca ttgaattcgt 24240 tttagttatt cacctttaag aagtagattc aaataaaaca gtaaccacat ttaattagag 24300 gataatagaa tgacacaaat agattttata gacatgcttc aaggaatagc agtattgctg 24360 ttttctattg gtagtgatac atttttttcc tatgttaaag atagagttat atctcagtct 24420 tctttttaaa ctttgctttg catcatttaa ttatttgcat catagttcta ctatgatagt 24480 tgggagtcca tagttttgag cagaagcagg caaatgatag tacctggtta cggtttagtt 24540 cttccatctt catagaggac agcttagatt tagtggtctt gttttaaaat aaggtccatt 24600 attttctctc accaagcaat tttgttccca atactaccgt ttcagaaatc agctttttca 24660 ttagaggaag ctggaggaaa aagagccata ccctttgcca tataagatat ttacagagtt 24720 taattgtatt ttatatgaaa aggagaatga aaacatatga ttaactatga tgctctatca 24780 tttttcttac attgaaagat cttaatatct tattagttca ggtaattgtt ttataataat 24840 cacaaatatg tcaacatacg tggagaaaat aaaaactctg tagttatttt aaaccaaggc 24900 cactgaggtg gaacaaaatg gctgaataga agcctccact gattgctccc cccaccaccc 24960 acaggaaacc aagttttaac aattatctgc acacagaaaa gcaccttcat aagaaccaaa 25020 aatcaggtga gcaatcacgt tacctggttt taacttcata tcactgaaag aagcattgaa 25080 aacaggaaaa acagtcttga aacgctgatg ctgcccctcc gcattcccca ataacaccca 25140 tgtggtgcag agggagaatc tgtacacgtc ggggagggac agcgtagtga ctgggggact 25200 ttgcattgaa ctcagtgctg ccctgttgca gcagaaagca aaactgttct gacctcagct 25260 ggcacctacc cacacagaga gcatttggac cagacatagc cagcagggaa gcacccatcc 25320 cagtggtcag aacttgaatt tctcagcagg ccgtgccact gtgggctaaa gtgctctgga 25380 gtcctaagtg aatttaaaag acagtctagg ttacaaggac tggaattctt aggcaagccc 25440 tgatgctgtc ctgggcttag agctggtgaa ctgggttaac atgtgaccta aggagacacc 25500 atgcagggtg gctgaggaag tgcttatgcc atgcttctcc caaccctaag cagtgtagct 25560 cacacccatg aaggtgactc cttccttctg cttgaggaga gtagggagga gagaaaagac 25620 ggctttgtct tgcatcttgg atactagctc agccacagta gggcagggca acgtgcaggg 25680 ttgtgaggcc cccatttcgg gccttatctc ccagatgaca tatgtagaca taccctgggc 25740 tggaagagaa cccactgcct tgaaggcctt gaagggaagg acacaatcct ggcaggattc 25800 atcacctgct gactaaagag cccttaggcc ctgaataacc aatagtgata tccaggtggt 25860 atgctatggg ccttaggtga gactctgata catgctggct ttgggtacca actcagccac 25920 actgggatag agaaccaggt gggaccttgg ggcccccaag tccaggccta ggctcttaga 25980 cagtatttct ggacctgcca tgggccagag gggagcctac tgccctgaag gctgagtccc 26040 aggcctgaca gtattcacca caagctgact gaagagtgct tgggccttaa atgaacattg 26100 gcagtggcct ggcagaactg cccatgggcc agtggtggca gcagcaggag aggctcttcc 26160 acttgtggaa agaggaggga agggtgggaa ggattttgtc ttgtggtttg ggttcaagct 26220 tagtagaata gaacaccaaa cctggagaaa gatgccaata ttcaagtaca aggttataga 26280 acaccaagca aatttaaccc aaagaagact acctcaaggc atttaataat caaacttcca 26340 aaggtcaaaa ataaaggaac ctaaaagcag caagagaaaa gaaacaaata gcacacaatg 26400 gagctccaac acatctgact gcaggctttt cagtgggaac cttacaggcc aggacaatgg 26460 catgacatat ttaaagtgct gaaagaaaaa acttgtaccc tagaatagta tatctcatga 26520 aaataccctt caaacatgaa atagcaagac agactttccc agacaaacaa aagctgagag 26580 atttcatcaa caccagacct atcttacagg aaatgccata gtttgcgaat ctgaaagaac 26640 aggatgagca ataataaatc atctgaaggc acaaaactca ctggtaataa taagcacaca 26700 taaaaataga ctattataat actgtttaat tgtggtatgt aaactactca tgtcttaagt 26760 agaaagacta aacgatgagc ccatcaaaaa taataaatgc aactactttt caagatataa 26820 acagtacaaa aagatataaa gagaaacaaa aaaagttaaa aagcaggagg atgaagtcaa 26880 agtgtagatt tttaaattag tctccttttt gcttatttat gcaatcagta ttgtcttcag 26940 tttaaaatca tgggttataa gatattattt tcaagcttca tggtaatcta acataaaaaa 27000 ctatatacaa cagataaaca aaaaatgaaa agtaagaaat taaaacatac taccagagaa 27060 aatcaccttc actaaaagga agataggaag gaaggaaagc aagagaagcc cacaaaacaa 27120 ccagaaaaca taaaacaaaa tggcagaagt aactccttac ttatcaataa taatattgaa 27180 tgtaaatgga ctaacttctc taatcaaaag atatagcatg gataaatggt tttttttaaa 27240 aaaacagcaa gacccagtgg tctgttgcct tcaagaaaca cactttacct ataaagtcac 27300 acatagactg aaaataaaag gttgaaaaaa tatattccat gccaatggga acaaaaaatt 27360 accaggagta acaatgcttg tatcaggcaa aatagacttc aagacaaaaa ctgtgagaag 27420 acacaaagta ttatataacg atacaaggat caattcagca agagggtatg acaattataa 27480 tttatatgca acaaacactg gagtgcccag atatatagag caaataatat tagagctaaa 27540 gagagaaata gacctcaata caataatagc tggagagtta aacacctcac ttttggcact 27600 ggacagatgt ccctgacaga aaaccaacaa agaaacatca gatttaatct atgctgtaga 27660 ccaaatgaac ataataaata tttacagaac atttcatcca acagctgcag aacgcgaatt 27720 cttctcctca gcatatgggt aatcctcaag gatagaccat atgttaggtc acaaaacatg 27780 gtttaaaaca ttttaaaaaa ggaaatgata tcaagcatct tctctgacca cggtggaata 27840 aaactagaaa ttaataacaa gaattttgga aactatacaa acacatagga attaagcaat 27900 gtgctcctga atgagcggtg gggtcaatga aaaaattaag aggaaatgga aaaatttctt 27960 gaaacaaatg ataatggaaa cacaacataa caaaacatat ggtatctagt gaaagcagtg 28020 ctaaggggga aatttatagc tataggtacc tacaccaaaa caaacacaca aacaaacaaa 28080 aaaatctaat tatgcacctt aaggaactag aaaagcaaga gtaaaccaaa cccaaaatca 28140 gtagagaaaa gaaataataa acaccagagc agaaatttaa aaattgcaat gaagaaaaca 28200 ataaaaatta atgatatgaa aagtttttga aaaaataaaa ttgataaacc tttaactaga 28260 ctacccaaga aaaaaagaga gaagacccaa atgagtaaaa tcgaaatgaa aaaggagaca 28320 ttacaactaa ttctgtagaa actctaaggc tcaatagtgg ctactgtgag caactgtatg 28380 ccaataaata ggaaaatctc gaggaaatgc ttaaattcct agatacacac aaagtaccaa 28440 gattgaacca tgcagcaatc caaaacctga acagaccaac aacaagtaag aagattgaat 28500 gcataataaa aaaatctccc agtaaagaaa aacccaggac ccaatggctt cactgctaaa 28560 ttctagccaa catttacaaa agaactaatg tcaatcctac tcaaactatt ctgaaaaata 28620 taggagagaa tacttccaga tttattctag gaggttagta ttaccttgat accaaaacta 28680 gacaaagaca catcaaaaaa agaaaacttt aggccaatat ccctaatgaa cattgaggca 28740 aaaatcctca acaaaatact ggcaaactga attcnnnnnn nnnnnnnnnn nnnnnnnnnn 28800 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 28860 nnnnnnnnnn nnnnatcaat ccttcttgcc tcctcatctc tataacaaca cttgataatt 28920 aaattgtgtc ttttctatgt gccaggcact gctttaagag ctttgcctgg gttaaattaa 28980 tcatcaggat gagactttga aattgttact actgatattc ccattttaca ggtgagcaaa 29040 ttcaactgca atttcctcaa taaccccagt ttaccatgaa tgcttatttt ctcagcttgt 29100 ataggagttg acagtctgtg gctcataaca gtcttccaga agttgaaata aattcaggtt 29160 cctacccgtg aggaccagtt aggatgcttt ttggtgaaat tttcactata tccaatgggg 29220 tttaaacaat aaagaattta ttatctctcc aaacaagaag tccaaaatta gggttgcagg 29280 gtggattgat ttagcagctc agtcatacca agatttcttt tactaggaag aaaatacttt 29340 ttccaaaccc ctaagcagtg tttctttttt tggcatgatc cctacgccat tcacaggcaa 29400 aggggaatgg atgatccatg actcctctct gagactgagc ctaactacca tgagcatatt 29460 gttgcctgat ccctgaacaa agcctggggt ctattagcaa aaggaaaagt gagggaacca 29520 aaggagcctt actctccagg acatctgcac gccaccagag tgcagaatgt aagctgtctt 29580 gcatgcgtat gaaaaacata actctaacac tgaccccaag gtctctcctg ctgtgcataa 29640 ctaataggcc ctgtgactgc ttactgctgt gatgcagaac agacttcaca ggtgctcctc 29700 ggctgggact ggtgtggcag cgtgggaggc aggggcatac tgtcactatg actgagcaga 29760 gtattattgg aaatggtgtt tgactccctt cttattggtg caactcaaaa gggcaatgta 29820 gtaaacagtt ccagacaaat ttgaagacaa gatgtttttc ataaggaagg aaaaaattct 29880 cacaacaagc caaatgtgca tcttttcaga taggagccct tcatattcac tggaatttct 29940 taaaaagtaa ctttccacat atttccccca aaataaccta tttcactaag agtaaagcac 30000 gtggcaccta atttatcttc cctgaacaga gtagagctga gtcactgaca cagaactgaa 30060 accttcgcac ccaagcaggc tcaagggact gagccagaac tgattctcag atagctcaac 30120 cctccctccc tctcccacaa taaaggttca tatcaaaatc acactctgaa catctgactc 30180 atatcttgtg attattgaac atactcctcc tgtaccaatg ttcctgtgga catttgacca 30240 tttccatgtt catttcttag ggaaaacagg attgtttagc tggaggttga atcaaataga 30300 tatacatatt gaaaccaagt gtcctctctt gctgtctgaa catccaaaca aagctaggag 30360 cagccccctt accattaatc aaggaccaga taaacagcta tctgctgaag aggacttctg 30420 cagcaatggc ttagccctca cctctggcac aaggaaggat ggatgggtgg gctgcaatta 30480 gttggcatgt tcaaaataag agacaagtgg cagggaacac ctcttggttt gacttcagct 30540 tgcatcactg tggctcttga ctgccatttc tacttttcct gtgtagatcg aaagctctaa 30600 tccttacccc tagtaagtgc aatagtgccc tttgtacagt tggagacctc cacctcctag 30660 tttgagccag aatgtgttct gtgcgtaaat acattacatg gagtccaaga gatttctttc 30720 tttcttaatt tacacataat aattgtacat attcatgggg tatgtaagga tgttttgata 30780 catataatgt atgatgatta gatcagggta attagcatat ccattatctc aaacatttat 30840 catttctttg tgttgggaac attcaatatc cttccttcta gctatttgaa actacatatt 30900 gttaactgta gtagtcctac agtggtatag aacactagaa acgtattcct cctcccttgc 30960 tctgattctg taacccttaa caaatctctc cctaaaggtt tctgttaact cctaataaat 31020 gatcgcgtgg gttggcagag tcaagaatga ggttaaatgg agaattcttg gcctgaggac 31080 tatgaattcc tttttttttt tttttttttt ttttggccca tttggttcca cgtgtgcctc 31140 cagatctctg gtgctgtgga gcagtgactg tgctgtgtgc tgcacacctc actctcttgc 31200 cgctttccct ggtctccatc aggactttac caaaacactc aagtgctgtg gcatctgaga 31260 atgtccctct atgcactgtc tgtccatata gtgagataag ggaggtgctg gaaacaattg 31320 ttctcttcct ctatgattcc tgcaagtatt gttttgcaat tgtgctgccc gccttgaaaa 31380 tcacatggtg ccccactggc agctttatga tcatttctct aaatgaaagt gagaggttat 31440 cgaatttgtc tagtttagtg tgagtcactt atatgcctgg gaaatgaagt cctatcctgt 31500 gttattgata ctggaagctg cacaaaagga atgagtctct ggagtttgga actcacctca 31560 tgaatctcac accaaccagt gctgtcctgg cttttcattg ggatggaatg agaatcaagt 31620 ccccatggtg gaggttatcg tgaaaacccc agattttaat agagaggtaa tttgagtaca 31680 taacatattg aacttgaata ttatgcactt gaaggaaaaa tatttccttt gtagtgggca 31740 gcctctatta tgttttggag gaattttcca gtaaatcatc taacagctgc tttactgtat 31800 aggccttgaa attttatctg ttcattggtt acagacactt ttgcttttgg aaacttcaca 31860 tgcttctctt taccagtaat attttgttcc caaaatatcg gctcctgggg aggatttaat 31920 aaaggctgac ccaacaagac atgtttttga gcaaaccttt tggatgtctt tggctaggaa 31980 ccatgtccta tgtaagtgta ttctatgtaa gattattact aaaaatccaa aaagcaacct 32040 acagattttg tgacaaaaag aaaatgttaa ccatctctga acataaaggc attttgcatc 32100 tgtgaatgta gaaacagtga ttatagtggg tgctagtaga ggtatagaat gtatgtgtgt 32160 gttttggagg agtggcaagg aagaaacaat gggacaagca ttggcaaaaa tatggctggt 32220 ggagattctt tccttcagac tgcagattga agacaccctt cacagacagg agccactgaa 32280 taaacacctc tgaagaagat agaaagagaa tagcttgcta atgccgtgct aactgaagtg 32340 agcctgcaat tctcaaaata tgatccattt gagggttacc tggaagatgc ttattccaga 32400 atagaggcat gatgtgttga tttttggaag gggctcctgg ccaacagcaa ttgaaaaact 32460 gaaaaaaatg gaggctgaaa actcaccaac catgacttca gttcatgggg cacagtcatt 32520 atctatgccc tttaagaaag aaagattcca catggacagt gtgtgaagac agtggagagg 32580 gtgaaggaag agcagaaagg tttgctctaa gaggcccatc tgacactggt cacaggtcag 32640 tataaggcag aagagaggag ggcaggaggc ttagcttgga gttggtaaat gaaagagttt 32700 tcaaggagat tttccagtca taaattagag agaacaagag cagaagaggg ggctatttat 32760 ggaaatattt gcaataggtg aatagtttgc aaattattgg ggaacctgca tttctttttg 32820 tctcatctat ttattttaag tcagaataat cccttatttt aaaaattata tgtattggca 32880 tattaaaaga ttaataagta aaatagttct ttccctctta ggttctgtag cactcaaacc 32940 aatggagaca aaaatatcag tttgattact gagaacaatt atattgcagg gtatgactat 33000 gtaagtggtc aaatagacat gctaatactc cacttcctca atctttgcct tgattcagcc 33060 caatcaaggt aagtttgggg tacttttacc catgattctt gtgtcacatt aatttgggac 33120 atggagagaa gaaaagagaa attcctagta attacaggtt taaaaagttt taaatagata 33180 agctctagtc aaatgaaacc aatagatctt acatagcaat tattagaggg tagaacaaca 33240 ggacaaaaga gttacctctc tgagcgaagc tcatctactt ttaaaaacct gtaactttca 33300 tatgacagca tgtgtagaaa attataataa tcatacacga tagtagaaat aaaaagctca 33360 tctactttta taaacctgta actgtcacat gacagcacat gtagaaaagt ataataatca 33420 tacacgatag tagaaaaaaa tggatgagtc agcttgaagt tagagaagtt agcttagggg 33480 ctctggcttt cacaagccct tcacggactt ggttcacttg taaccaaagg ttgtggtaca 33540 ctatgtacat agatacggga tctgggcaag ctgattggca aggtcagccc taaggatctg 33600 ccacctgttt tcttattgta ctttttgtga tgatcacgta atttttcttt tttaatttgt 33660 taaccagtga agtgtctttc caataattcc ttgattctct gacagcaact gggtgtccta 33720 taatgtagtt cagttctgac acaacccaga atgagtgcag attccacagg ttaagggctc 33780 agtcccccaa gaccaccctc atttcagaca ctggtggtaa gacctggagg ttatgcatac 33840 ttctgactga ccagctatcc atttggggtt ccctatgacc tccgctcagg tttaataagt 33900 cactagaatg actcacagag cttaggaaag ctttataatt atgattatgg ttttattata 33960 aagaatacaa ttcagaaaca gccaaatgga agacaggagt agggccaggt attgagtgtg 34020 tgtaggtggg gccagggaag acacagagct tacatgtgct ctgtgtgcac catcatccca 34080 gcacattgat gtgttccccc acctggaggc ttccagagcc tcttgttcaa agtgtttaat 34140 caaggtttca ttacatacac atgattgact aaatcattgg ccagtggcga ttcaacaatc 34200 tccagcctcc ctctcctccc caaaagtggg gcggctgagt gggtgagtgt gtaaggctga 34260 aagttgtaac gctcagatcc caagctttgt ctttctgatg ataagctccc atcctgaagc 34320 tatctagggg ccctgccttg agtcacctca gcatgaactc aggtatgatg gaaacaagct 34380 cgttatgaat aacaaaagac actctgtccc tcaggaaaat ccaagggttt taggagctct 34440 gtgctagtaa tctgagacaa aggcaaataa gttttttatt atgcagtaat aaaagggtaa 34500 attatattca tatattttta aaaatcttaa acggttcttt cattctttaa ctgcttatat 34560 tttatatact atttgatttg gtatactaat accttgtttt ggattttcac atagtaaaaa 34620 tggcctatag ttttccttgt tgatattgtc attgtctaat tttgttagca aggttcaatt 34680 gggcttatta aattagtttg ggatattttc tctttttcta gtcttaaaaa gagtttaaga 34740 atatgtatgt tccttgaatg tttcattgaa ttcatatata gaactttctg agctggagtt 34800 tacttttgag aagtctctta attaccatgt taaaagaaaa actttaaaca aattaaattt 34860 aatgggattt aatgcagcaa agaataattc acaaatcaga aagccctgaa ccagaacagg 34920 ttcagagaga ctccggtgct gctgtttggt tgaaaaatat ttacggacag taaaaggaaa 34980 gttatataca gaaaatggaa gagaggtaca gaaacaccca gattgattac agcttggcag 35040 ttgaacacaa gagcaggtta caatttgttt acccatccag ttaggttaca gtttactatg 35100 tacagagaaa cctttacgct gaacttaaaa tacataagga ggcagattca gttaaacttt 35160 taaaaacaac cgattcaata gcaaaaatga ttataagagc attcagatgt tctatttatt 35220 cttgagtctg ttttggtaag ttatgttttc taagaatttg ttcatatcat taggttttta 35280 aacatgtgga cacaaagtta tttatatttt cttatgtctc aatgccaact gcaattatat 35340 ttatgcccct cttttgaatc ttcacacatt tcatttttgc ccaagtacca aggcagaagc 35400 catagcttat aattcaatgg aattcttaat gcgagccctg acaaaagtat tttcccttta 35460 gatactttgt gtgtgtgtgt gtgtgtgtgt gtgagtgtgt gtgtgtgtgt gtgtgtgtgt 35520 gtgtgtgaga tagagagaga gagagacaga gacagagtca ggctggagtg cagtggtgcc 35580 gtcctggctc actgccgcct cctgggttca agtgattctt gtgcctcagc ctcccgagta 35640 gctgggacta aaggcacgca ccatcacacc cagctaactt ttgtattttt tagtagagac 35700 aggttttcac atgttggcga ggctggtctc gaacacctga cctcaagtga tttgcccacc 35760 tcaaccttcc aaagtgctgg gattacaggc gtgagccacc gtgcccggcc tccccttaga 35820 tacttatgaa tgatattaat ttctgccatc tgcccccata ctgtctccct aacccaggat 35880 gtaatactgg ttttgataat ccttcttggc tggggatgtg gaaagttcta gacgcttcca 35940 cctggccatc tacactatga tagcccaatg tgaggttatg ctaactgcca agtgtgccaa 36000 tcccaaatat tcagtaggag aaaataaacc tgcctataga aatagaacaa gaaataaccc 36060 tgatggtgaa attagtagat aggattttag aaccactatg ataaagatat tcaaatattt 36120 atttatttgc atgctaataa ttttttattg gatgccagaa tttgtggatt gtacctttgg 36180 ggtgctgaat attctttttc ttttatatta tgctttaagt tctagggtac atgtgtacaa 36240 catgcaggtt tgttacatat gtatatatgt gccatgttgg tgtgctgcac ccattaactc 36300 gttatttaca ttaggtatat ctccgaatgc tatccctact cccttccccc accccacaac 36360 aggccccggt gtgtgatgtt ccccttcctt tgtccacgtg ttctcatttt tcaatcccca 36420 cctatgagtg agagcatgca gtgtttggtt ttctgtcctt gcaatagttt gctgagaatg 36480 atggtttcca gcttcatcca tgtccctaca aaggacatga actcatcctt ttttatggct 36540 gcatagtatt ccatggtgta tatgtgccac attttcttaa tccagtctat cattgttgga 36600 catttgggtt ggttccacat ctttgctatt gtgaatagtg tcttaataaa cataaatgtg 36660 catgtgtcct tatggtagca tgatttataa tcctttgggt atatgcccag caatgggatg 36720 gctgggtcaa ttggtagttc tagttctaga tccttgagga atcgccacac tgtcttccac 36780 aatggttgaa ctagtttaca gtcccaccaa cagttaaaag cgttcctatt tctccacatc 36840 ctctccagca cctgttgttt cctgactttt taatgattac cattctaact ggtgtgagat 36900 ggtatctcat tgtggttttg atttgcattt ctctgatggc cagtgatgat gagcattttt 36960 taatgtgtct gttggctgca taaatgtctt cttttgaaaa gtgtctgttc ataccctttg 37020 cccacttttt gatggggttg attttttctt gtaaatttgt ttaagttctt tgtagattct 37080 ggatattagc cctttgtcgg ataggtagat tgtaaaaatt ttctcccatt ctgtaggttg 37140 cctgttcact ctgatggtag tttcttttgc tgtgcagaag ctctttagtt taattagatc 37200 ccatttgttt gggttttgtt gccattgctt ttggtgtttt agttatgaag tccttgccca 37260 tgcctttgtc ctgaatggta ttgcctaggt tttcttctag ggtttttatg gttttacgtc 37320 taacatttaa gtctttaatc catcttgaat taatttttgt ataagatgta aggaagggat 37380 ccagtttcag ctttctacat atggctagcc agttttccca gcaccattta tgaaataggg 37440 aatcctttcc ctatttcttg ttttctgtca ggtttgtcaa atatcagatg gttgtagaga 37500 tgtggtatta tttctagggc ctctactctg ttccattggt ctatatctct gttttggtac 37560 cagtaccatg ctgttttggt tactgtagcc ttgtagtata gtttgaagtc aggtagtgtg 37620 atgcctccag ctttgttctt ttggcttagg attgtcttgg caatgtgggc tcttttttgg 37680 ctccatatga actttaatag ttttttccaa ttctgtgaag aaagtcatta gtagcttgat 37740 ggggatggta ttgaatctat aaattacctt gggcagtatg gccattttca caatattgtt 37800 tcttcctatc catgagcatg gaatgttctt gcatttgttt gtgtcctctt ttattttgtt 37860 gagcagtgat ttgtagttct ccttaaagag gtccttcaca tcccttgtaa gttggattcc 37920 taggtatttt attctctttg aagcaattgt gaatggaagt tcacccatga tttggctctc 37980 tgtttgtctg ttattggtgt agaggaatgc ttgtgatttt tgcacattga ttttgtatcc 38040 tgagattttg ctgaagttgc ttatcagctt aaggagattt tgggccgaga cgatgggatt 38100 ttctagatat acaatcatgt catctgcaaa cagggacaat ttgactttct cttttcctaa 38160 ttgaataccc tttatttctt tctcttgcct gattgccctg gccagaactt ccaacactat 38220 gttgaatagg agtggtgaga gagggcatcc ctgtcctgtg ccagttttca aagggaatgc 38280 ttccagtttt tgcccattca gtatgatatt ggctgtgggt ttgtcataaa taggtcttat 38340 tattttgaga tacgtcccat caatacctag tttattgaga gtttttagca tgaagtgctg 38400 ttgaattttg ttgtaggcct tttctgcatc tatcgagata atcatgtggt tttcgtcttt 38460 gattctgttt atatgatgga ttacattttt tgatttgcat atgttaaacc agacttgcat 38520 cccagggatg aagcccactt gatcatggtg gataagcttt ttgatgtgct gctggattcg 38580 gtttgccagt attttattga ggatttttgc atcgatgttc atcagggata ttggtctaaa 38640 attctctttt tttgttgtgt ctctgccagg ctttggtatc aggatgatgc tggcctcata 38700 aaatgagtta gggaggattc cctctttttc tattgattga aatagtttca gaaggaatgg 38760 taccagctcg tctttgtacc actggtagaa ttcagctgtg aatctgtctg gtcctggact 38820 tttattggtt ggtaagctat tattgtctca atttcagagc ctgttattgg cctattcaga 38880 gattcaactt cttcctggtt tagtcttggg agggtgtatg tgtcgaggaa tttatccatt 38940 tcttctagat tttctagttt atttgcatag aggtgtttat agtattctct gatggtagtt 39000 tgtatttctg tgggatcggt ggtgatatcc cctttatcat tttttattgc atctatttga 39060 ttcttctctc ttttctttat tagtcttgct agtggtctat caattttgtt gatattttca 39120 aaaaactagc tcctggattc attgattttc gaagggtttt tgtgtctcta tttccttcag 39180 ttctgctctg atcttagtta tttcttgcct tctgctagct tttgaatgtg tttgctcttg 39240 cttctctagt tcttttaatt gtgatgttag gatgtcaatt ttcgatcttt cctgctttct 39300 cttgtgggca tttagtgcta taaatttccc tctacacact gctttgaatg tgtcccagag 39360 attctggtat gttgtgtctt tgttcttgtt tgtttcaaag aacatcttta tttctgcctt 39420 catttcgtta tgtacccagt agtcattcag gagcaggttg ttgagtttcc atgtagttga 39480 gtggttttga gtgagtttct taatcctgag ttctagtttg attgcactgt ggtctgagag 39540 atagtttgtt ataatttctg ttcttttaca tttgctgagg agtgctttac ttccaactat 39600 gtggtcaatt ttggaatagg tgtggtgtga tgctgaaaag aatgtatatt ctgttgatct 39660 ggggtggaga gttctgtaga tgtctattag gtccgcttgg tgcagagctg agttcaattc 39720 ctggatatcc ttgttaactt tctgtctcgt tgatctgtct aatgttgaca gtggggtgtt 39780 aaagtctccc attagtattg tgtgggagtc taagtctcgt tgtaggtctc taaggacttg 39840 ctttatgaat cttggtgctc ctgtattggg tgcatatata tttaggatag ttagctcttc 39900 ttgttgaatt gatcccttta ccattatgta atggccttct ttgtctcttc tgatctttgt 39960 tggtttaaag tctgttttat cagagactag gattgcaatc cctgcttttt tgttttgttt 40020 tgttttccat ttgcttggta aatcttcctc catcccttta ttttgagcct atgtgtgtct 40080 ctgcacatga gatgggtttc ctgaatacag cacactgatg ggtcttgact ctttatccaa 40140 tttgccagtc tatgtctttt aattggagca tttagcccat ttacatttaa ggttaatatt 40200 gttatgtgtg aatttgatcc tgtcattatg atgttagctg gttattttcc ttgttagttg 40260 atgcagtttc ttcctagcat caatggtctt tacaatttgg catgtttttg cagtggctgg 40320 taccggttgt tcctttccat gtttagtgct tccttcagga gctcttttag ggcaggcctg 40380 gtgatgataa aatctctcag catttgtttt tctgtaaagg attttatttt tccttcactt 40440 atgaagctta gtttggctgg atatgaaatt ctgggtttaa aattctttct ttaagaatgt 40500 tgaatattgg cccctacgct cttctggctt gtagagtttc tgccgaggga tccactgtta 40560 gtctgatggg cttccctttg tgggtaaccc aacctttctc tctggctgcc cttaacattg 40620 tttccttcat ttcaactttg gtgtatctga caattatgtg tcttggagtt gctctttttg 40680 aggagtatct ttgtggtgtt ctctgtattt cctgaatttg aatgttgcct gcctcactag 40740 gttggggaag ttctcctgga taatatcctg aagagtgttt tccaacttgg ttccattctc 40800 cctgtcactt tcaggtacac caatctgacg tagatttggt tttttcacat agtcccataa 40860 ttcttggagg ctttgttcat ttctttttac tcttttttgt ctaaacttct cttcttgctt 40920 catttcattc atttgatctt gaatcactga taccctttct tccacttgat caaatcagct 40980 actgaggctt gtgcatgcat cacgtagttc tcgtgccatg gttttcagct ccatcaggtc 41040 atttaaggag ttctctacac tggttattct agttagctat tcatctaatc ttttttccaa 41100 ggtttttagc ttctttgcga tgggttcgaa catcctcctt tagctcagag aagtttgtta 41160 ttatcgattg tctgaagctg tcttctctca actcatcaaa gtcattctcc atccagcttt 41220 gttccattgc tggtgaggag ctgtgttcct ttggaggaga agaggtgctc tgatttttag 41280 aattttcagc ttttctgctc tagtttctcc ccatctttgt ggttttatct acctttggtc 41340 tttgatgatg gtgatgtaca gatggggttt tggtgtggat gtcctttctg tttgttagtt 41400 ttccttctaa cagtcaggac cctcagctgc aggtctgttg gagtttgctg gaggtccact 41460 ccagacccta tttgcctggg tatcaccagc ggaggctgca caacagcaaa tactgcagaa 41520 cggcagatgt tgctgcctga tccttcctct ggaagcttca tcacagaggg gcacccggct 41580 gtatgaggta tcagtcggcc cctactggga ggtgtctccc agttaggcta cttgggggtc 41640 agggacccac ttgaggaggc agtctgtccg ttctcagatc tcagactcca tgctgggaga 41700 accactactc tcttcaaagg tatcagacag ggacgtttaa gtctgcagaa gtttctgctg 41760 ccttttgttc agctatgccc tgcccccaga ggtggagtcc acagaggcag gcaggtctcc 41820 ttgagctgtg gtgggctcca cccagttcga gcttcctggc agcttttgtt tacctactca 41880 agcctcagca atggtggaca cccctccccc agccttgctg ctgccttgca gttttatctc 41940 agactgctgt gctagcaatg agtgaggctc tgtgggtgtg ggaccctcca agccatgcac 42000 gggatataat ctcctggtgt gctaaggcca ttggaaaagt gcagtattcg ggtgagagtg 42060 tcccaatttt ccaggtacca tctgtcacgg cttccctttg ctaggaaagg gaattcccta 42120 accccttgtg cttctcaggt gaggcgatgt cccgccctgc tccgtgggct gcacccactg 42180 tctgacaagc cccagtgaga tgaacccagt acctcagttg gaaatgcaga aatcacttgt 42240 cttctgtgtc gctcatgctg ggagctgcag actggagctg ttcctattcg gccatcttgg 42300 aacctcaaga tattcaaata tttaaagaaa aacatgaaca tattgagaaa acagagtatt 42360 tcaacagaga aatgaaatct ttaaaaaaag aagcaaatag aaattataat actgaaaaat 42420 atggcatata aaatgaaaat atcacaagat aggtttacca ttactgaagc aaaaacttga 42480 agacaggcca caagaactat ggaaaactga agcagagaag ggaaagattg aaaaaaaagt 42540 gaatgaagcc tcagcgaccc atgaaacata tgaagtctct ctctctctct ctctctctct 42600 ctctctctct ctctatatat atatatatat atatatatat atatatatga tagatagata 42660 atcaggagga aggagaagga acagattgga gcagagaaac atttaaagaa atattggtcc 42720 aatattttcc aaattgaatg aaaaatataa tgtaataaaa ttgatgaata tgaagcaaga 42780 tgaacacaaa actgcatcat cttataatca aactgctaaa agtcgatgat aaagagaaaa 42840 tattaaaagc aaccagaaaa aaggcacatt acgtagaggt aaacaatatc aagaataatt 42900 gctgacttct gagaaaagaa atacagctca gagcagtctg agctctgtga ggtgtgcaca 42960 attcgtcagg cccagagaga caggagtatg ggacttcagc cacacacctc aatcccttcc 43020 tgcatccatg tgcaggcatg actgtttaaa ggtatttttt tcccgactgg gcatggtggc 43080 atgtttgtgc ctgttgtcct aactactcag gaggctgagg cacagggatc acttgagccc 43140 atgaggtcaa ggctgcagtg agctatgatt gcaccactgt acttcagtct gggcacagag 43200 ccagaccttg tctctaaaaa atatatatat atatataacc acttgtaatt gctgtaactg 43260 ctgctaacca gagcatatat ttgagcaact tgaatcccta ggctcctggg ttgcagtgct 43320 caaacttggc ttaaataaac tctttattaa tttgtcctca gttttttcct tttagattga 43380 cacttctcag taacaataca agccagaact aatggatact tttggcccat cacttggacc 43440 acttcaatcc atgtgtatct cttcaggtat gctgtatgat gttcttacag acagaatctg 43500 tatagtgcta tttctagcca gcctccaacc ccaggaatct ggcttaaata aatccaagta 43560 gtaaagccag gaacatatca tatgttatgg aaattatgca tcaggtggaa aaatatctta 43620 gagttgtggg aatactttga tatatgttct tattaatcat caatgtgcta tataacagaa 43680 tgcatgctgc tttaaatttt aaaaccgttt gtggagaaaa aaaaaagaac ttaccacctg 43740 gctgggtgca gtggctcaca cctgcaatcc caacactttg ggaggccgag gcagggggat 43800 cacctgaggt caggagttcg agaccacctg accaatatgg tgaaaccttg tctctactaa 43860 aaatacaaaa attagccagg tgtggtggca tgcgactgta gtcccagcta ctcgggaggc 43920 tgaggcagga gaattgcttg aacccaggaa gtggaggttg cagtgagccg agatcgtgcc 43980 attgcactcc agcctgggca acaaagtgag actccgtctc aaaaacaaac aaacaaacaa 44040 aacaacaaca acaaaaaaac tattcgtggc taataatcta atgtgttcag aagagaaacc 44100 ctagtacaag aaaaagaatt ctgtgtgaag aactatgtga agaactgtgt gaagaaacta 44160 tgatatagaa agtcagaaga tattgtacat tacttgttct aaagcatcct gagaaagcaa 44220 tattaagatc tcagaagtaa gtaccaagaa aaaaatgatg ttggtttcaa aagtagtaaa 44280 agaaatgagt ttaggcctgc catggtggct cacacctgta atctcagcat tttgggagac 44340 taaggcagga gaattgctta aggtcaggag ttagagacca gcccgggcaa tacagggaga 44400 cctcatctct acaaaaaata aaaatattag ctgggcatgg tggcacatgc ctgtgatccc 44460 agctactcag gaggtggagg tcggagaatc acttgagccc aggaagtcga ggttgcaatg 44520 agctatgatc acaccactgc actgcagcct gagcggcaga gtgagaagcc tgtcttgggg 44580 gaaaaggaaa acgaaaacaa aatgagttta ttcaggctgc agaagttcaa gtcaaaggaa 44640 agcttagaaa ttttatttac atcaagataa agaagtttaa agcatatgat tattaaatat 44700 ttcgttggaa gatagcattt aatgacagag aaaatagtta cgatattatt atatagaaat 44760 aacaagtcac acattagtaa tggatgtggt atcatccagt aatagagaaa gtaggcttaa 44820 aggtaatttt tactttcttc tttttgcttt tttgtatacc attatgcatt ttctacaata 44880 aacatttttt taggtttgta cattgcttaa aaatgtttgt tagactttat tattttaaga 44940 ggagttttag ctttacagca aaattgagca gatgggtaca cagacttcca tatgcaccac 45000 cacctcctcc cacccgcagg catggctccc aattatcaac actgcccaca gagttgcatg 45060 tttgttacaa ctgatcaacc tatactggca tatcatgatc acccaaattt tataaacatg 45120 ttattatttt taatatattt tatcttttga acagttttag gtttacagaa aaattaagtg 45180 gaaagtacag agagttccca tattcccctt cacttccccc accccagatt ccattatcaa 45240 taatactagt attagtgtca tatagttgtt acagtaatga gcaaatattg atacattatt 45300 aactaaagtc tgtagcttac attaaggttt acactttgtg ttgtacattc tatggttttg 45360 acaaatgtat attgaactat atccattgtt acagtatcaa ttcattattt ttgtaattag 45420 aaaaatggtt ataaacacaa aattaatgat tattacatag atgatagtga tagtatctca 45480 ggctatagag ataagctatt gcttaattcc actgaaaaca gaatagctct gggagttcat 45540 ttctatttat tgcttctaat aataatacca atggcttaac ctagaaaaat attatctttc 45600 cctattaatt tatgtatgta ccctttgggt ctgagaaggc aattagtttc attgaggtca 45660 ttcttaaaaa aaaaaataga acaattggaa gatttttaca tcttttcaat tacatgtcta 45720 aaactatgcc aggcacattt gggggtctct agtgcctggg atgatgagag aagaccttgt 45780 acagaagatg atctttgaac tagaacttaa agaataagta aaaatggaaa aagaaaacaa 45840 taaaaggaaa aggagagtag catccatgca aactgtaatg gttaatacat tacattagag 45900 ggttatactg tgtgaattct tgttaaaaaa gaatagatct ttgttggatg tttgagtaaa 45960 agcttaatag acggagtgcc tttaagttgg gttgtgataa aattagacaa agaaaaatac 46020 agaggaaaag taaaagtaaa ctcagccgcc tgggtgtggt ggttcatgcc tataatccca 46080 gcactttggg aggccgaggt gggcagatcc cttaaggtca ggagttcaag accagcctgg 46140 ccaacatggt gaaacccatc tctactaaaa atacaaaaat tagcttggtg tcatggcagg 46200 tgcctctagt cccagctact agggatgctg aggcaggaga atcacttgaa cccgggaagt 46260 ggaggttgca gtgagccaag atcatgctac tgcattccag cctgtgtaac agagtgagac 46320 tctgtctcga aaaaaaaata aaataaataa aataaattca gtcaaaatgt ggaggatcaa 46380 aatctgagga gtgattctat gataagtaat acagagagtg catatagaag aggtttgtgg 46440 gagataaagc ttggttggtg acaaggaagg acttgatgga tagtcttccg agacggggtt 46500 tgatcacagc aaacccgaga tggagtcatt ctgtattata ttaacgtttt ctgcatttaa 46560 atctgtatat tggatagact ggggacaaaa aaaaaagaga caaagaccca tacaattatc 46620 taggcgtcca gagcctacat ttaaaaagtg acataggact agaaagaaag gatgatctgt 46680 aagacatttc tatgaaaaag tagtatcagg aactaatgac ttgtattagt ctgttttttc 46740 catgttgctg ataaagacat attcaggact gtgaagtaaa agagtttaat aggacttgca 46800 gttccgcatg gttcgggagg cttcagaatc atggcaggag gtgaaaagga nnnnnnnnnn 46860 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 46920 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn agttttgtaa tgacctatct ccagagtttc 46980 tcacacatca tcattttagt acttgagaat gcctccatct tcctgtaact aaatctacag 47040 acctacttac gatcacacct tcattgttct tctatccttc atctacaagc agttggggac 47100 actatcgccc ctacgatcac aggccaatcc ctccatctcc tttcacttta ttgaagacag 47160 tgcttttctg ataatcctcc ttcttccttg aattagcaat gcctcccttt atgctacaga 47220 actcccatca acattcaaat aaagaatgtt tacttttatt tttgttttaa tgttatattt 47280 aatcaacatt tgctgttaat ggcagcatgg ttttgttttt gttttttgtt tgtttgacac 47340 agagtctcat tccatcaccc agggtgtagt gcagtggcgc aatctcggct cactgcaacc 47400 tccacctcct gggttcaagc cattctcctg cctaagcctc ccgagtagct aggattacag 47460 gcgtgtgcca ccacactggc taatttttgt attttgagta cagatggggt ttcgctacgt 47520 tagccaggct ggtctcgaac tcctgacctc aggtgatcca cccacctcag cctcccaaag 47580 tgctgggatt acaggcataa gccaccacgc ctggccaggt tggttctttt cagacacttt 47640 cctcctagga tctacagttt gcttctatta tccactacgg aaggcatgtg ctgatactgt 47700 ggttggatcg ttaattcttg ccatcatgtg acctgctgcc actagaactg cagaatgttt 47760 ctaactagtt caacatattg ggtatcaggg agtggcttcc tctgattccc aggttgcagg 47820 aatttcttaa ttctggggat gttgctgatt cctgtttaaa acgccttcaa cagagggaag 47880 tcagaaagaa cagaagcatt gagttcttct actattagaa cagcttctaa cagttgtatg 47940 tctgcccaac tgaatttgtt gccaacaaga agatcctctc catggtcttt caaaatcttt 48000 tcaaagatgg ccaagtactg ggttttagct ttcttcacaa ctgagcaagg ttctcctctt 48060 tttcctcagt gggccagcgc catcatcatc atcaggtcca gggtgccctc agcacacatg 48120 ttgatcccga ctctctcctt caggtccttt ccacagacat tgtacttagc agcaagatag 48180 ctgaggatgg ctgtagtcag tgtcagcatc attccatcaa tttcaaccaa aggcacttgg 48240 ccaaaatgca ggcgtctatc cttctgcaac ttttcatatt attatcttgt ttcaataaat 48300 tcttcttcaa actctattcc agctgcagcc agcagccaga ggactgactc caccctgccc 48360 ctgacatgaa agtagtagag cttgggtttg gctgccatgc ctcctggctc aagattttct 48420 gttcagctat ctggaagctc caagaatgtt tatttttaaa aagaaaggag gccaggcgcg 48480 gtggctcatg cctgtaatcg cagcactttg ggaggctgag gcaggcggat cataacatca 48540 ggaatttgag accagcctgg ccaatatggt gaaatcccat ctgtaccaaa aatacaaaaa 48600 ttagctgggc gtagtggcgg gtgcctgtag tcctagctac tcaggaggct gaggcaggag 48660 aatggcgtga accttgggag gtggaggatg cagtgagcca agatcgtgcc actgcactcc 48720 agcctaggtg acagagtgag actccgtctc aaaataaata aataaataaa taaataaata 48780 aataataaat aataaataaa taataaaaag aaaaacaaaa ccttcaattg tgcttttcac 48840 tccaattatc acccaattaa tttatctgtt tcctgtacca acattgtctg tgcagactat 48900 ctatatttac tcacgtctta ttctctttaa aattaggaaa tatttcaaac atacagaaaa 48960 tagcatcatg aacacttatg tatcttcaac ccaactcttt gccatcctgg aattttgtgt 49020 ttatcattcc tatgcaaatt tttactacac ttattactac atatgattgt caaacaatct 49080 aagatattgt tactacatga ttttaaattt tatataaatg ccatcacaca atttgtatcc 49140 ttctgcaaca tgctttttaa tattacattt ttgaaatgta tccccattaa tacgtttctc 49200 aatgattttc acagctgtat agtactctgt gagcatagta caatgtatcc attccattct 49260 ctagcgaatg aacatttaac ttatttccaa tgtttctcta ttacaaaaga tgtctcagcg 49320 aactccttgg gcatgtattt tagtataaac acacacagtt cttaggcatg tatctcatat 49380 atacatacat atgtgtgtat atgtaacata aatatatatg tgcatctctg ttttgtccct 49440 ttaattccat tggtttatta tccataaaaa aatttgtatt atattgccta atttactatg 49500 attcctgata ggtcttgata tctgtagggc aattccctcc actttacctt tcttaaaaat 49560 tatcgtaaac aatcttgacc ttttttctac catgtgaaat ttaggatcta cactgcatga 49620 aattttttag gacaatgata gaaacacatt gaatactgac tcttctctct tttgaacaca 49680 atgtatttct ttacttattc agattctggg agcatatcct tcagaaacat tttgtagtag 49740 tcttcataag ggtgtcatat gtctttttta gacttattct taggtataaa tattttaaat 49800 gaaaaattca tcataggaac tcaacaactg ttaatccaac tgatttatgg acagggaact 49860 atgaagattg catccattta taataatgtc tgtgatggtt gtgctctgcc tcagatcaat 49920 tctttaccct cttcttcctg ctctgtgcct gggaggctga attctacaga tggacatcat 49980 ctgtgccttc agttctctga cttctaatag gaattatcca aaaaaggcac caggaggaga 50040 ctgaagagca ggagggcaga aagtttagga tatttgtttc ctagtcttcc ctgttccctg 50100 ttgttctgat agtggctgaa ttcttccaga cctcagctcc tgtcaggaag ctaccattcc 50160 aagcgtctag cactcatcag ggtctgagaa taatgttcag tttcaatgac ccatcaggcc 50220 tagccatggt aatggcatcc tactgtccta ctgctgttaa tccttgagtg tctcttcatc 50280 cctcgtggtt ctcttaacct tacctttcat gcttccaaag gaaaactaca ctctaaatta 50340 gcataaatga aattctcaag attagttttt aaccttacca acacctctac aaatggtctt 50400 tttataaatg ttcttcagtt aagtcctttt gagtatgcca tctgttttct gttgggatcc 50460 tgactaatat aattttacat tctaaaatta aaattgctaa ggagtctggg tcttccttaa 50520 atagcctggc tttgcatttt gtttttactt cactgatttt cacctacttg actgctggaa 50580 gagatgtcat aaacctggga tagcccaagt attaaccctc ctgatcaaat gactagaatc 50640 tataaatgtg tctggttaaa tcaacaataa cctctcaaaa tttcatagta ctgatttaaa 50700 tatagttgtc agttgtcaca cgtttgagtt ctggttgtta aagttatcac catgctgttg 50760 gtctcatatc caactttttt cattctttcc cctttttaag caatgacatt tcaaaaacat 50820 gatgaaggaa aatggaatga gcatcacatc atataaacac aaattacttg attccataac 50880 tagaaaaagt gctggggtag aaaaagactg acagcttttt cctcttctct gcccacaatc 50940 tgtagtatat ctgtggggag cttcctggtc ttcggatatt tgtgccttta aacctctcaa 51000 agtttttcaa ctgatactaa aaatgccttt taaagtttta actcatttta aaaatgaaat 51060 gagcaattgc aaaagattta catttcttct aaatgcctaa tttaagagaa aagcccagac 51120 tgctattcct gaaaaattcc atgtcactct caaccttcca ctaatgagat gaatatgtta 51180 aaatctcctt cagcaaatac gattttacca cctgctcact ttctcaaact gaatgaatat 51240 acaagtaaat gtcacataaa aaatggcatt tccagaagtt taatccattc tgtttgtttt 51300 aggttaaaac ataaaaactt tcatccttcc aaaggaaagc tacactctaa attagcataa 51360 atgaaattct caagattagt ttttaaatta caaatcaacc aaaacagtga tagagggccc 51420 tctgctgttc aaaaatagta agtggaaaat ggaaatcatg tccattcttt taagatcccg 51480 ctgcctcttc aataaatcac aacctttctg gagggggagt gagagactag ggtgtcattt 51540 cccaacatca cttttataaa gatgaagcaa aatttagaac ttaacggctg gtgggttttt 51600 tgtggatact ttttagttgt agatgggtaa gttaggggaa ttgtgaaaaa cagctattga 51660 gaaataccat tggaaatgtg gtaaaagtgt attttaaact ctaacaacgt aggatatggt 51720 tcaagatctg gccgaagact aacttgggca acaagcaaag aagctgctca atatataata 51780 atggtgacag taacaaatgt tattttttaa atctgattac attaatatca tgtagaaaaa 51840 aactagtaag atctattaaa caattgctca gtgtttttgg ttacttgtat ctgaactttt 51900 tgttttttgc ttgtcattta ctcttttatt attaatacca atactaatta tactttagca 51960 tatgaatcag tgtccatttc ttagttctta aattatactt atttttttaa aaaggttcaa 52020 accaaatttt cttgagcatt tcaggtatta attacttatt tgatgaacta accaatcagg 52080 atcttcacat agttctgtta gtaatgtaca actcaatgga taggagacta aatccgtttt 52140 aatactgttt ccacaataaa ttctaacact accgaaaaaa gcattataaa gcaataaaaa 52200 agaaataatt accgtttaat attgtttaaa agctaaatga aatttgcagg cttaaaaaca 52260 actgtttttc ttccaggagg tttatagagt ccttttcctt attcatcact acgtagaaag 52320 ctagagttat tttctagcta gccagaataa tgaccacgtt taatatcttc ccattctgtg 52380 ctgatattaa gacgactgac cattgttctg cctttctgtt agttgctctt aagttcaata 52440 aagaaagaat tcttagaaga aaaaggagag aaagacagaa aggaaaaaaa cgagaaaaaa 52500 agagaaagaa aagcagctat tgcaaagtgc atcttttatc acataccatg gaatatcatc 52560 ttaagaagtg ccatatgtca cttaccaagg cctttcatgt atagtataat attgtatagt 52620 tatggtatgg cannnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 52680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnttgaggca 52740 cccttagaga tgcacagaat tctaatattc ttaggttaaa cgttgccagg ggctctaaca 52800 tgggtaacac gtaatggtta gcctatgcaa cttacataat gacattaaca agagtacttc 52860 taaagttttc tcgttcttta tgtggagatt tgccctttga tttggaagta gatggtccaa 52920 cataactatc atcaacttgg tggggtaatt tgcccttttt tccaaaacgt tccatgtatt 52980 cagcttaaat taaaacaaaa aaaaaagcac aattattcat ctaaaaggta agctaaagca 53040 tttcacatgc ttaaaataag atgctaaatc accttttaat aaaaaattac attatcaagt 53100 aatcctaagt ataatttttt ttttttacta tcaggaggtt aaactactcc tcttttgtat 53160 catcataact agaaacatct atttcaagaa tattccactg atttatattt tgattaatat 53220 actaataaaa ttttctatta tatttacata tgattctcta aaagtgaaca ttaaaattat 53280 gataaaaaca ggatacaaaa ctatttgaga aattttaaag aaccatgaca aaaagttaat 53340 attaactgta atcaatctga taaacactac atttaatgag tcaaaataat aaaaacatta 53400 aaatgactaa tttggaagtc atgttcacaa tttattaaaa cccacaaaaa attttaaaaa 53460 gaaatataat ttattaaaat ctctcttata aaaatctcag tctttgatgt agaggcaaat 53520 tctgaccaca ataaataaac gttttagtat caacaaccca attcctaaaa tatcagctct 53580 tatagcattt acaacagcct aaaataactg agaactacaa tcaattattt tctgagggaa 53640 gatgtattgg gaaaattttt ttctacttga cattaatgtc attcaggttt tgtacttcta 53700 ttacactatt aactaaatat tgtctggtct ctctctacaa attatcctta aatctaattt 53760 agaatattga agttcttacc atgggactgt tcatttttaa cactaaatgg ttctggactc 53820 tcatcatcct gctttacact caaatggaat gatggagctg cctgctgcca gtggggcttt 53880 gtactcacct aaaatacaaa atttaaatag cattaacaaa caatatcaga aaaaaacctt 53940 tttccatgaa atgcacgagt gtttataaac acagacattt tctcaaacag atcagaacat 54000 ctggatagaa aatactcaac agtagatctt attcaagcct gtgaatggtc atggtgtgca 54060 ccatttgggg gacattaaca aaagtcctgc tgttggttgg atgtgaatgc ctgttaccta 54120 aaaccagggt cttcattcta gagaggctac tcctcatccc cagatattgt caagtgtctt 54180 taaaccttct attttaatga gaagtgctct ctgctggtct cctctgcatt ccagctccaa 54240 aaggtcttcc tgttctcatg gctgtaaacc accctagatt gctctgacaa ctcacaaatg 54300 tattccattg tttcaggcag gggtgacaaa ttattataca tacacaattt tggcaggtgt 54360 cataaaaagg gagaaggcta atgatgatag gttcctggca ctcaggctcc atgttgggaa 54420 acaacagtgt gtggtgagga ctgtggcaga ggagagagca ccttcatctt ctaaaaacgc 54480 aatgactaat tagctcaagc caattattgc aataccagaa cacaaaccta ctgttgctga 54540 agcctatggt tcttcaacaa acccagacag tcggatttta aagtaaaatc tacatatttt 54600 tagcacaatt tagtctgtct ctccctttca aatccaaacc aacttaaaag tccatgtctg 54660 tactggcttc tctttctgcc tagttttact ctccctaatc tcttacttcc acttccctaa 54720 taaactacct gtacacaagt ccttatttta gttacctctt acacaggaaa ataggataag 54780 aaaacatgta agacagtgct cttccatttt ccacccgccc ttcccatttc tcaactctcg 54840 tcttgctcca taaaagtttt aaaactcata gaagcgctac gaggagttgt acagaaaggg 54900 tgagaatcag gtcacagagg ctgggagcca ccagtgccag atcattcttg atttcaacta 54960 caggggtcac ctctagaagc ctgaagtctg taaagaaaat cactccaggt cgtaattact 55020 gctggagaac ttctatccag gtacaaacct taatactgta ttcaaataaa agtagtttta 55080 aaaaaataat ccatagctca ttttctaaat atctgttgaa aatatgattt cccttaaaac 55140 ccataagact caaagaatgc tgaggaaaaa ttgcaagaca cagccaaata tgaatacaaa 55200 accaggttta catcagtcaa taaaagcatt ttagatgatt atatgcttcg cccacaagca 55260 agaattatta tgcgtctcag ccatagatat tcgataagaa aatttaaatt aagtttatat 55320 ttctacatct gtaaactaac aatctgcaac aaactaaaaa atgtctattc catttcagtt 55380 ttttatcttt tacactagaa aattaagagg gattttgcag aactagagaa aacatgctat 55440 ggctcgattc tgcaggcatt ttacaataat ttgccattat caataaaagc atattttcaa 55500 ttaagatttg tatttttaaa tgttcaatat aattaatcta acaatgttag ttcaagtttc 55560 ctaagcgaaa ctgtccttca tttcaaatta tcagtaatat tctctgtaca aagcaacaca 55620 aattcaattc tatagtttgt tctcagcttt taaaatatct ttcaaagcaa ataatgcttt 55680 ggatgaaccc tgaagacatt atgttaagtg aaataagcca ggcataaaag gacaaatatt 55740 gtatgattcc accaatatga aatacctaga gtagtcaaat acatagagac aagaagtaga 55800 atggtggttt gccaggaggt gggggagggg aaaatgggga gtcattgttt aacggacagg 55860 gttgctgttt agtcagatga aaagagttct ggagatgagt ggtagtaata attatacaac 55920 aatgtgaatg tactgaatgt cactgaatac ttaaaaatgg ttaaaatagt aaattttatg 55980 ttatgtatat tttatcaatt tttttaaaaa ataataatat ttgcattgac cctcccctta 56040 aatttaactt tcaggagtca caagactgta acttggtggc attaaactcc acataattac 56100 tagacaataa attttattac attaaaatct aacttgtaca gggctttatc aactgaaaat 56160 gtctattaac tgcctcttcc acaagacaat gacactttat agtcaaaata atgaaaaaag 56220 attccaaaat ctaagaaata ttttgtagta cagcattttt ctgctggaag acccatctcc 56280 ttgaagatca tcaaatcaac tcagctcatt ttcaaactca gaaactgagg ctcaagaggt 56340 aagtgactcc ccaaagggca cagagccatt agaggcaaag ctttacccca agcccttttt 56400 tatttactcc aacgctaata ctccttttgc taaccaatct tggcaagtcg ttcattcaga 56460 atactgaaat attctacata tacaggtaat tatagcttat ggttacttta ctaagatagt 56520 tatttgtcct aatatgcaca cctcatagaa tttctccatc agaaaagaat caaatggagc 56580 aggaatttgc ttacttattt tcaagttaga aaactaataa acattttttg aagcacaaaa 56640 attcaatgag aatatatttg gataagttac catagacagc tgtggtaaag ctcttgctgg 56700 tgccttgaac ttttctttgc tggctaattt ctcctgtgtg aaaaatatga gggggaaaaa 56760 agtgtttact ctgttagtgc taaaattgat tcactgaacc aaattttaca gaaagactat 56820 ttaaaaagta catattatcc aacatattca aagataaact ttactgcttt ttaaattctc 56880 aggtttgtct cagaattgtt ttctttagag caagaaaata aaggaaaatt atatccagca 56940 atacttatca gaaagcttta aaaagtatgc aaaaccggcc aggcgcggtg gctgtaatcc 57000 cagcactctg ggaggctgag gtgggcggat cacaaggtca ggagatcgag accatcctgg 57060 ctaacatggt gaaaccccgt ctctactaaa aatacaaaaa attagccggg cgtggtggtg 57120 ggtgcctgta gtcccagcta cttgggagac tgaggcagga gaatggcgtg aacccaggag 57180 gcggaggttg cagtgagcag agatcgcacc actgcactcc agcctgggcg acagagcaac 57240 actctgtctc aagaaaaaaa aataaataaa aaataacaag ttctacctag gaactactga 57300 cactcaccaa tcaaaactcg ccagctcttg taagacactg ccagtgccaa taaactttct 57360 ttcagaacaa cttgtataac ctcctctttc cccaattaaa ccctaatcat ttaacttgtt 57420 ctccagacat actggaggcc accctagtct gtacgtaagt cctggattgc aatctcactt 57480 cctgtatatt attctcaaat aaaacctttt tacttgtatg cttatattgc aagttgacag 57540 caactttcca caagtctcac ttataccaca gacagagact tcctttcaaa ctttcaggat 57600 cccagtaggt ggttttcacc tcccagccca gacctgcagc tcttcagcaa atgtctttgt 57660 catccagtgg gccacagcca catctcccat gaggtctgaa tctaaggctt tggggaaggg 57720 acggctcttc caagtttgtt ccttgagcac tctctctcag acctagaggg agtggctgct 57780 ccttatatct gctattcctc tattcttcag ggctgtgctg tccaaaacag tatgcactag 57840 ccccctgtaa ctaccaaaca cttgaaatgc ggctcctctg aagtgagatg tgtttaagtt 57900 taaaatatgt actagatgtt gaagacttag tacaaaacac aaagaatgta aattatcaat 57960 gtttatatat tgattagagc atattttatg ttgattacca tatgtgttcc acattgttct 58020 aagaagctta catgaattaa cacctttaat attctatacg ctgtggggta aatactgttg 58080 taccactttt gtgcctatta cgtagataag aaatcatgta gtctctgcct tcacaaagca 58140 cacagtccag ttaatattaa agaccatgtg cttccatcta ggacaggatg tggagcagag 58200 tattctaaga acgtttcaaa aagaaaaagg gtggagctaa gccttaaagc atacctggat 58260 ctccacagag aaggacagca aagagcttac tgaagtcaga gggcgcatca agaattggag 58320 tctaaatgtc aatggcaggc tttgaggcta tgttggagtg gatggggaag gtaatccatt 58380 ccctaactca acaatggttc accagctcct accatgtgct gggctcatag actaagtcat 58440 aggactacag actaagatac ctagtccata gtcgtggaaa ggactaagtc cctctgctta 58500 tggagtacac attctagagg aggtacagac agttatttca gaacagatct ataacataat 58560 ctcagaaact agtatgtaca atgaagaata ataaaacagt gtgaaggaac agagagggaa 58620 ggagtggaaa agtgctaggt agatggagta gtaagaccct ctctgcagag gttatgtttg 58680 aataggaacc tgaataaaag gtaggggaag aacaaactag agctagacaa gtgggtgctg 58740 ttatattaag gaggacatgg gtttcagggc ttgactggcc aggctggcaa tagagagcca 58800 ctgcagtctc cttttagggt gaactttagt ctcagtttgc ggaagacagt ctcagtttac 58860 acctttagtc tcagtttgcc caagcataac tagtagaact ttcgctctca atggcatctt 58920 agtttagatg ataaattata atgctatcct gtttatgatg tagagacata caaaagctat 58980 ttcataaatg gttctgatga tacaaacagg atacatgaga agcaggggac tagagtcagc 59040 agtgaggcta gtttagcagg tttttaagaa taatataggc aacaggagac atgggcctgg 59100 atttacagaa aatgctggta acaaaaggtc acctctacct ctcaaaggca ctgctctgat 59160 catactgctc ccctgttcag aaacatttta aagccccctg ttgaagtagg cacaactctt 59220 cttggtgtcc aaagccttcc tcaacatact ccaaactatt ttccagcttc tcttagttac 59280 tttgtagcca tatgatttgt gtggctagtg aaatgtgagc acaacaggaa acacagttaa 59340 gtgctagaag gacaggcagt taagagctag agggacaggg agttaagagc tggtgtgctt 59400 cctccatcac tatctcacac ccatgaagcc catgtgttcc cactggcata gctataagat 59460 gcagcgctac ctgaccctcg tcagacttca tgtgagcaag gcctaatctt tgttgggtta 59520 agtctctgag atttcgactt ccatttgttt tgtcagatag tattaattac cctgtcagag 59580 ccaccaatag gttccaaaat atctccctgt taaaatctag ccctttaaga ccaaaatcga 59640 aaaagtttac atttcatttc ttaactccaa ctgtaagtaa tctcctttga agatgcatag 59700 cattctttat tgtttaaatt atttttggct gggtgtgatg gctcatgcct gtaatcctag 59760 cactttggga ggcaaagggg agcggagtgc ttgagctcag gagttcaaga ccagcctggg 59820 caacatggtg aaactccatc tctaccaaaa cagatacaca aaaataacct gggtatggtg 59880 gtgcctgtgg tcccggttac tcaggaggct gaggagggag gattgcttga gcacaggagg 59940 tggaggttgc catgagccaa gactgcacta ctgcactcca gcctgggcaa cagagagaca 60000 cctgtcttta aataaataaa atatattctg atatatgaat tatgtgtaca cctacctata 60060 ctgttctcct aacccctcac cccaccccac ctccacctac taaatcagtc ttatagagat 60120 gagtgacatg gtttggctgt gtcccaaccc aaatctcatc ttgaattata gttcccataa 60180 tccctatgtg tcatgggagg gacctgggtg ggaggtaatt gaatcatggg agcagtttcc 60240 cccatgctag tcttgtgata gtgagttctc acaaaatctg atggttttat aaggggcttc 60300 cccttcactt ggctctcatt cttctccctc ttgtctcctg ccaccatgtg aagaagaaag 60360 ttgcttccct ttccacgatg tttgtaagtt tcctgaggcc tccccagcca tgtggaactg 60420 tgagtcaact aaagctcttc tcttcacaaa ttacccagtt tcgggtattt cttcatagca 60480 gcatgagaag gaactaatac aatgggaaaa ctgactttaa agtcatttta gctttatctt 60540 agaaaatcat aaaatggtga ggctgggcac aatggctcac agctgtaatc ccagcacttt 60600 tggaaagcca aggcaggagg atcactagag cccaggagtt caagaccagc ctgggcaaca 60660 tggtgaaacc ctgtctctac caaaaataca aaaattagcc aggcgtggtg acccatgcct 60720 gtggtcccag ctactggtga ggctcacata agcccaggag gtagaggcta tagtgagcca 60780 tgatcacacc actgcactcc agcctggaca acacagcaag acctccttgt ctcaaaaaaa 60840 aattacaaaa tggttaacca tgccctaaaa aatggataac taaagataag aaaagaaaag 60900 aaaggaaaag aaaaggaaag agtgcaatga gcttcagaca gtactttctg aaatacaagc 60960 accagattag gggaacgttt cacacatata caatgctgta acccaaatgg ttctggtttc 61020 tacaattccc actgggctac acaatgggaa gggaaatatg agcacaacat tttcaagcta 61080 acaggtgcaa taggtgttct cagagttgaa gccttccctt tggggagaat gagggtgtgg 61140 caaaggcctg agaagaatgc agttatagat gacagagagg aatggctctg ggatgtctag 61200 tccaagtgcc ctggaatttt cacatcaggg acagaggtaa ttcgaatgac atctattgtc 61260 ctggagtcac tacaaatatc caaaacaccc tatgtaaccc aagcctccta tacaacaacc 61320 ccatcactgc caccaccctg actttttaag atctctgcat catccatgct gttgctgagc 61380 tatttctcca agtctcacca tcactctagg tgaagaaggg tggtgaggac cttaatgttt 61440 tcttcttaca ttgcagtgcg gaatatgtat cttatagaat attttacaaa tagtgcttct 61500 ctagctaacc aagacaagca gccacacata tgtcagaaaa atcatctggc aaatgatgcc 61560 tcacatcctg ctggcctcac cttggattcc agacatggct gcagtaaaca gttctgtgct 61620 agcttctcac atcaagaaaa tatcttttgt ggccgggcgc agtggttcac gcctgtaatc 61680 ccagcacttt gggaggccga ggcgggcgga ttacctgagg tcaggggttc gagaccagcc 61740 tggccaacat ggtgaaacca catctctact aaaaatacaa aaattaggca ggcgtggtgg 61800 catacacctg taatcccagc tactcaggag gctgagacag gagaattgct tgagcccggg 61860 aggcggaggt tgcagtgagc caagatcatg ccactgcact ctgtctgaaa aaaaaaaaaa 61920 aaagaaaaaa gaaatgtttt ttcttttctg cagggggaat cctctaatgc tacagtatgg 61980 gactctagga agagcccact ctgctcacac acatgagcag ccagaagagg atgggagtca 62040 acatccccag gaacaatctt tcaccaatac aggatgaaag atgacagata aatgctattc 62100 cttgaccctc ctcaggtgat caattccagg aagcattcta taagctcaga ggtcatggca 62160 ggacatacca gttgcctact gtgctaatca acttgatagc ccatggttgt gtggcctttc 62220 cttcttccta cttttactct cctcagtctc ttattattgc gtctcaaata cactacctgc 62280 acacaagtcc ttgtttcaga taacttgtgc acaggaaaat aggctaggaa aacatgtaag 62340 tcagtgctct ttcatctttc catttgctct ttcattgttc aactctaatc ttgttccata 62400 aaagatttaa gacttgtggg agggatatga atgaggtgtg cagaaagggt gagaatcagg 62460 tcaccagctc acaggggctg agagccacca gtgccagttt caactacagg gttcacctct 62520 agaagcccga aggtccgtaa ttaaaatcat tctatgtctt aattactgcg gaagaactta 62580 tagccaggaa caaactttaa tattcaaata aaaattttta aataaatgaa tgtttttaaa 62640 tacctcttaa aaatctaatt tcttttaaaa gacataaaac tcaaaggata cggaaggaaa 62700 attacaagag ccagtcaaac atgagcacaa tataaggttt taaatcagcc aacgaaagca 62760 ttttagacaa ttacatgcct tgctgactag aagagattat tcaactcagc cactgaatat 62820 ttattcagca agtaaattta aatttaacac gccaaaaaag ctaatacctt ttttcaattt 62880 tagaatttgt caaatttttt aaaagagaaa atgtctattt catttcaggt ttacttataa 62940 actagaaaca agataaatat ttgcagaact aaaaaaaatg ctttgattct attttgtaga 63000 acttttggta atcttggtat tattaatatg aataagtaca caattaaaac atgtacttga 63060 aatgtccaag atcatacact taaaattttc atccaagttt ttttatttca aaacattagg 63120 aatcaactct gcataaagca gtccaaattc cattacacag ttcatgctga gctcttaaaa 63180 tgtttcaaaa caattaatat ttgaattgaa actccactta aactcaactt ctgtagaagt 63240 tataatagtt taactcaacc gcattaaact caacatacct agacagcaaa ttttattctg 63300 taaaatctaa cctggaccca accatcctgt acatacctga aaattacagc ctatagttat 63360 tttattgtta ttattcttcc tatgtacctc acagaattcc tccatcagaa aagaatcaaa 63420 tggaatttaa aattgtataa aatttaaaat cagaaattca gtaacttact ttcaagcttg 63480 aaaagtagta aacaaacatt atcataaagg aatgaagtat gatggtatat tggtaaatta 63540 ccatagacag ctgtggtaga aatcttactg gtttcttgga tttttctttg ctggccgatt 63600 tatcctgtat gaaaaacatg aaaaaacaaa actgtttact ccgttaatgc taaaattgat 63660 tcactcaacc aaattttaca gtgtttattg taagcataca tcatccaaaa tattctaaaa 63720 catactgtac aaatggcatg atcttatata ctgaaaatct taaataattc atcaaaaaat 63780 attaaagcta gtcaatgagt tcttgtatcc aggtaggttg caggatacaa gatcaacaca 63840 caaacatcaa atatatatag gatacgccag gagggcgatg catcccaatt ccacagggag 63900 aggacatgga agctccgcac ccaggactct cccaggccta tccccatgtg tctctccacg 63960 tgttttttcc tgatttgtat cccttataat aaaacagtaa taatacatat tctaaaaatc 64020 aattgtattt aatataattg tatttaatac aaaattgaaa ttgagacaat tccatttaca 64080 attgcatgaa atagaataaa agacttagga ataaatttaa caaaagaagt gtaagaccta 64140 tacaatgaaa ccataaaaca ttattgaaaa aaattaaagt tcttgataaa taaaaggaca 64200 acccatgttc atggattggg agactaatat tgttaagatg caatacaccc tgaaaatatt 64260 ctacagatgg aacataatcc ctatcaaaat cccagcttcc cttcttgtag aaattaacaa 64320 attgatcata aaattaacat ggaaatgtat caagaagaat caaacaatct tgaaaaagag 64380 gaacaaattg gcagactcac acttcgagat tttacgactt actacaaaac tacagtaata 64440 aagactgcat tgctctagca taaggataaa cagagatcaa gggaatagaa ttaggagttc 64500 agaaataaac ccagacattt atgggcaatt gatttttcat gagggttcca agacaattta 64560 atggagaatt gggcatatct tctggaagtc tgtctgaatc taatttattc aaaacttact 64620 tggccccacc tcccttctct ataactaaaa cttcaatttt ttttttccta gaatggtcta 64680 cttcctaagg tgaatatgta ttctggtttt tccccccgca agctgccata agtaacattt 64740 gaacctctta gcagtaacct taagagaaag gggctaatct cctttgcagt agagaataat 64800 tccatggtag aagttttgtc taagtgagag gaagcatttg ttgaaaatga caatttgatc 64860 tataaagcag ataatatgag ttgtgcttct ggccagttgt caatttattg cctcagctcc 64920 aaatctgcct tttttgccct cctttgggat atgtcacagg acattataac atttctcctt 64980 tgacaaccag catattatca tgggtgccaa gaagcactgt aagaggaaga ggctttcttc 65040 ctgattccag tgctttttct catagctccc atggtgtggc tgccagcagt cagcaacctt 65100 gtcaatctgt ggtgtgaggc taactcagag gcagacaccc tccagcaagt ttcgccagca 65160 tccctgtggg cagctttcca caagtttcac ttacatcaca gagggagact tcctttcaaa 65220 ctttcaggat cccagcaggt agttttctcc tcctagccca gacctgcatc tcctcagcaa 65280 atgtttgtcc acccactggg ccacagccac accttctccc atgaggtctt aaatctaaag 65340 gctttgggga agggatggct cttctcttcg agcagagagt ctctgctctc ttaacattct 65400 ggttaatatt aaaactatgt ttctggctgg gcgcagtggc tcatgcctgt aatcccagca 65460 ctttgggagg ccgaggtggg cggatcacga ggtcaggaga tcgaaaccat cctggctaac 65520 acggtgaaac cccgtccgta ctaaaaatac aaaaaacaat tagccgggtg tggtggcagg 65580 cgcctgtagt cctagctact caggaggctg aggcaggaga atggtgtgaa cccaggaggc 65640 agagcttgca gtgagccaag attgagccac tgcactccag cctgggcaac agagagagac 65700 actgtctcaa aaaaaagaaa aacaaaaaca aacaaacaaa aaaactacgt ttccattcaa 65760 taaagagaga tggcttggag cagagtattc aaaatatcta cttgcagtct gtccattatc 65820 ccccaaactc tcttttccaa aataatgagg tttattttca atgtatatgt cctccattca 65880 cagatagtct cttttctgtc tcagtctaaa gattttctgc ttatctctgg tgttgtttcc 65940 cttatagagg ccaaggtggt atttactttt atttctatca cttggccttt aatacatttt 66000 tcagtcaact actaaaggac attgtttttg ctgcagcaat ttgaaactct ctctaatgtg 66060 ccttttgttc tctattattt tcactagtat gcaaacatgt taaaatccca tcagaaaaag 66120 tatcagcctt aatcctatat tccctctagc aattgcccga tttttctgct cccaatttct 66180 ccagaatgtt ctccagtgag gtatttattc cttgcatacc actctcatca agagctctga 66240 ttacctccac aatgccaagt cctgtggtaa attctcaggc ctcattttac tcaatctttt 66300 aacagtattt tgacacagtt gatcactgtc tccttcctga atactgtctt cactcaattt 66360 ccagaatgat tatatatttt atactcttcc taactctgtg aacacttaaa cttttcattg 66420 ccaattcatt ctcatttttt aaagttcatt tttaaattta caaataaaaa ttgtatacat 66480 ttatggtata caatgtggtg ttttgataca tgtataaatc gtagaatggc taaatcaagc 66540 taattaacat atgcattacc ccacatactt attttttgtg gtacgagcac ttaaaatcta 66600 ctctaatagc agttttcaag gatatatttt tatgaagtct agtcatcatg atgtataata 66660 gatctcttga tcttatttct cctgttcctt ctcattttct gatcttcgtg tgacagacat 66720 ccctcaatcc tcaggtatgt ttcctttcta attgacccca tccagcccaa atattttaaa 66780 tattacccca tatactggaa tctcccaaat ttatctcccc agtattaacc tttatcctaa 66840 gttccccact cctatatttg atttttgctc tctctactca gaggtcaaat agacatctct 66900 aacttaatgt gtccaaaata ttgtttgatc aataaactcc cacaaaccct accagaactt 66960 ccccatattt tcttaaactc agtaaatggc atcaccatta acaccattat ttaagctaaa 67020 atccctggta ccatccttga ttcctccctt tttctcatat cacacattta attcagtagt 67080 aaaccctttg agctttacct agaacatata tcctggatcc aacatctcac aatatccatt 67140 attactgtaa ccatggtcta aggaactgga aagaaagtgg agagtggaaa gagagatcgg 67200 ggtggagaaa aagaaagaat gggggatcaa gaaagaacgg gaagaggaaa ggagacagaa 67260 tccttattat atcatgtgtc cattacatac ctgaatctat tctgatgtcc attctcttgg 67320 atttcccagt tcagtaaacc ataaaaaatc tgttttattt tttgaagttt gagttccatc 67380 gtacttactt acaataaaaa gagcttctac tcttggtcat cagtctcagt gacttaaaaa 67440 tatacatgtg actaaatcta cctaacagcc tagtctctta gttacttaac ctcctcatct 67500 gtaaccttct cttccacttc atctctgtca atgactttca tggcaacccc tctaagatct 67560 tatcactaaa tatggcagct cttgtgaaat cttgctttta aaacattctt ttctctgacc 67620 accacatcct ttgctcccag caagtatgat atgatgccct cctatatttc tttgttttga 67680 ctgagacctc taatatctaa gctccttctc agtatccatc tgctccctca tatcttctct 67740 tcctccttgc ccaatttaga ttctacagtc catagtcaca actacaccct atcaaatacc 67800 ctcaactccc tctggcacat acttaactaa accataatcc cattggaatt caactactta 67860 ccttctctat gcttgtaccc aagtaggtat gtattactga ggaaaatcac atctccagac 67920 ctattggatt cactttaagt gcatgattag taatctcaaa cattgaaaac ttttgagatt 67980 ttctaataaa nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 68040 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn aaaaaaaaaa 68100 aaaaaacaac ctcttggtaa tacctggaac aattagataa gaagtgttat ttacagataa 68160 aataaataat aaaattattg cagaactgaa ttgtcacaag cactactata aactgttttt 68220 tcagatgcac agttttcttg gctaactagt ttctgtttcc taaattttac aagtgaattt 68280 ctttataaag cccaaaattt ccttcctgtt ttagtcaagt ttttgcttcg aaatctaagt 68340 aagaattgat agaagcaagt gagttacact tctttaacaa aactgttttt ttgttttttt 68400 tttttttgag acacagtctt gctctgtccc ccaggctgga gtgcagtgac gtggtctcag 68460 ctcactgcaa cctccgtctg ccgagttcaa gtgattctcc tgcctcagct tcctgagtag 68520 ctgggattac aggcatgtgc caccaagccc ggctaatttt ttgtattttt agtagagacg 68580 ggatttcata gtgttagtca agatggtctt gatctcctga cctctggatc tgcccgcctt 68640 ggcctcccca agtgctggta ttacaggcat gagccaccac gtctggccca acaaaactat 68700 tttaaaggga ggaattgttt tatagtgatt agaacagaaa gttaggaatt cacatttcac 68760 agttttattc ttgaggctcc gattccattt atgagctttc atgaggtatt taatcttatt 68820 ctgactcaat ttatccagta taaaatttat gaaacaccag tggattgaat ataaagggac 68880 taatttttgt gtgtgtgtgc tcatcgaaac ccatcaattt gtattccttc tgcatacaat 68940 gagaaaatat tttatagtta agcattctca aatcttaaaa ctgctttcaa agaatcaggt 69000 ttttatatgc aaatttattt agttgatgta ttaatatatt ttgataaatg tagaagagtc 69060 aaattatagt ttgatttcca gtactttcca ctgacatcaa ttttgaggaa atttgggtaa 69120 taaaaagtag gactctttgt catcttggcc tttgcgaata gctagtaaaa tacagtcaag 69180 cagtgtagtc ccccagtttc caccccgtac cccctaaaca ccaagttttg ttttccatgc 69240 taagagtctg atagactgtg ttagaaatat attgaaggga attgaggcaa gaggagttat 69300 ggaacaaaca ttgcttccta aagatctttt atttatacct gatttgttag tatcagaatc 69360 cagagagtaa ttcaaaattt aaggtagagg ttcttaattt tggatcccct atactcagcc 69420 ttataaggtt tatggatggg ctttaagatc tgaaaaccct ctggaattat atgtaaaact 69480 tcctatttaa gggcttgtgt atatttttct gggtaggcag tcaatagaat agcttttatt 69540 aatttcccaa aataatcctt gactcagaat agctttaggc ttttgtgaaa ataaattttt 69600 ggccgctgta gacttacagt aacacgtatg ccaatagaaa tgaattttcg gtgtctttaa 69660 attttggcag aggtactaag cactgccatt ggagagagct aaaagcatac ttatttctaa 69720 aatgataaac atgaccttaa tgggtgtgtt tgtacatagc tgtgataacc attattttct 69780 tagtttatca tgaagtatct ttgaatattt tctgtaggac acatatacct tagttatctg 69840 ttacaggctt caaagactac tgagcattac taagagacat attgctaaaa caattgacaa 69900 ttgtgatagt ttcaaaggtg gtaaaaattg tgttgaaagg aaaaattact aataaaatta 69960 aataagtcaa atacccattt ttgcattcac tttaatttct tatatttaaa ggtaagctat 70020 tctaattaga gagttaacat gataccctat gaagggtaca gtttttggaa atagacctga 70080 gtttaaatct ttaccttgac taaattacat atcttctctg aggtgtgaag aacaggagag 70140 catttattat ataggattgt tgtgatactt aaacacaata atttatatca agagcatggc 70200 atggagttgg tgtgcaaatg agattatttc cctgcaatga taatgtatct tttttttatt 70260 ttttttttga gacagtctca ctctgtcacc caggctggag tacagtggca tgatcttggc 70320 tcactgcaac ttctgcctcc cagattcaat agattctcct gtgtagctgg gattacaggc 70380 atgcgccaca acgcctggct gatttttata tttttagtag agacagggtt ttgccatgtt 70440 ggccaggctg gtcttgagct cctgacctca agtgatctgc ctgcctcggc ctcccaagat 70500 aatgtatcat tttagtagtg aagaataaat ataattctgt cttatcctgt tgttatagtt 70560 tgccatttac atttgtacgt gatagcacta taaattttat aaaagtacgt atgatatttg 70620 aatcaattta gagaacaaag ataaattcct aatgacacag atatttcaaa gaattgactt 70680 taagttggag gtataacaga atctgtaata tactaacgtt gttctctgca gaaaaattaa 70740 aattgattgt tctcatcttc ccccagagga aaatttgctg tggttagaca atgtatatca 70800 aaatctactg gccaagaata tgctgcaaaa tttctaaaaa agagaagaag aggacaggat 70860 tgtcgagcag aaattttaca cgagattgct gtgcttgaat tggcaaagtc ttgtccccgt 70920 gttattaatc ttcatgaggt ctatgaaaat acaagtgaaa tcattttgat attggaatag 70980 taagtatata ctaaatagta cctattttag ttggtatgaa aagatgatta aaatgtcaag 71040 aataagaatg ataaaggtgt gacaagaact tgtaaagatt cctgttaata aaatgtaaaa 71100 gggacctaat ttatattcag atattttcat aagactaaat gtatagcctc gggcacatac 71160 acaaaaacta acagcacaga ataagcaaat gacaaataca acatttgagg aatgtggaga 71220 aatgaatata cattattttc ttaatggcta attgattgaa tttttctgga acacaatctg 71280 atgggcagat agaattgaag taatttgtat attttcttca tctatttgaa aaataccttc 71340 aagaaaataa gaatagaaag gagaattgaa gtaatttgta tattttcttc atctatttga 71400 aaaatacctt caagaaaata aggagaatag aaaggtaaac tgcataaaac tttgcttaat 71460 acattgtttg ttaaagaaat acatttggta tatgtaatgt gtaaagcact atgttagttg 71520 tagcaataat aaatatatct cttgccacaa gtctgtgaaa aagtgaaaac acccccagta 71580 ttctacagtt caccaggtgg taggggtaga ggattgttaa ggtaaccggt ttgttaccta 71640 gtatattgac atgtgactct tacagagtgc ctttgttgca tgattgtctg tcacgtttat 71700 tctaaggatg taaaatgtgt ttgaacatct gcaacaactg gaagagatca gctagattta 71760 tgtttagtat accttgggtg ccaatagttt ttaatgaagt aaatatattt tatgataaac 71820 caagaagaga taagtgttta ttttagtaaa aaatgtattg ctaactatag taattctttt 71880 tagtaactaa cttgcatcat tgccttttta aatttaaagg tatattaacc ttatttttgt 71940 atttagttat tttaaaatta atgtttttgt tccacgaaat agattggctg gaatcaactt 72000 gtgaatgaaa atgaagattt tgcaaaatgt taaactgaca agtagttcat ttaagaatga 72060 atactatgta catttcccag aaccgtctta aggcgtgtta tatagcactt tcaaaataaa 72120 ttagaattgc agaaaaggcc aagatactac caatatgatt tcagttttca tatattttgt 72180 aattattttt gtcaaagatt cttcagctga cctttaaaaa gttacctact tgcataacat 72240 ctgcatgttt gaagtgtgta aaaagtagct aacatttgtc aaaacagtag tggaatacta 72300 ggtttggagg atgaatatac ataagaacat tgactatttt cataaaatcc tcttttgggg 72360 gttcatccaa acagcaacga caaaatacat cggaaagatt tgagacattt tatggatgga 72420 ccttgttcat actccggaaa attgactcag gtaccttacc cagtaaactg gcagaactta 72480 ctgatctcta tacctctggc tgaaatgtag caagcactca gaaaatgttt aagtagatta 72540 atgtattaca tttcttacca gagtgactaa gacccactaa aatttattgc tagaacttcg 72600 gcttaattac tatgccttcc gcttttgggg tggcattgtc acatttagta gtctcagttg 72660 aaggtaaaaa tttaatcagg tttagttgtt ttagtagtgt ggcataattt ttatatggtg 72720 aaattaaaac ggtggaatct ttaataacta tcacccagtc aattctgtcc tcaaatcatc 72780 ctacacactt tttcttcccc aactcttatt ttacaaaatt ccaaatctat agaaaagttg 72840 aaaaactagt acaattaaca tccttatact cttcactttt actcaccagt tgttggcatt 72900 ttgccacatt tactgaacca tttgaaagtt gtaggtatta tgatattcct ttttgtgctg 72960 aagtatttgg tgtgggtgat gatgtgtatt tattgtatca catcaggtgg caataaagtc 73020 aggttttatt actggttttg ctaaatttga acatgcttaa ggtgtagtct tctagctctc 73080 ctcattgtaa aggaacattt tcccttttta attaatctgt gtagtgatat ttacagacca 73140 tgtgattatt atcttttgtt gttgttgttg ttgttgttgt tgttgttgtt ggagtcttgc 73200 tctgtcacca ggctcgagtg cagtggcgcg atctcagctt actgcaacct ctgcctcctg 73260 ggttcaagcg attgccctgc ctcagcctcc tgagtagctg ggattacagg cacgtgccac 73320 catgcccagc aattttttgt atgttactag agatggggtt tcaccatgtt ggccaatatg 73380 tcttgatctc ctgacctcgt gatccaccca cctcggcctc ccaaagtgct gggattacag 73440 gtgtgagcca ccgctcctgg ccaatattat cttcttaact tttcgtctag tgatttccct 73500 ttcccacttt ttaatatttc tgtggatttc atgtttttta aattaatcta acacatggca 73560 ttaatggaaa ataacccatt atgttttaat taatgagttc taatcagttt ctgccactgt 73620 ctttttttca tgctcaaatt gtcccaagct tatcagtggt tccctttcaa gctacttcct 73680 atgtcctttt gatataatgt cattggtgtt tgaagactcc cttgctttat gcacaccaaa 73740 atttccaggg tcatcttgtt atcttgcctg ccccagacct gtaatcagcc atctctctga 73800 gaagttctgg ttcttttaag tggggaatag catttagaaa ccaaatatgg gtgttggaca 73860 tgctcacttt taacggagca tcgttatttt taggactttt ctaaggatag tgttaggaaa 73920 tcatattttt tcaaaatcac gaatttagtc tcatagctcc aattcaagtc caccactaca 73980 aggttcttca tcacttctca cattctatat atgcatctcc cctctctcac agtgaaaacc 74040 caggttctca gcaatacttg cttattcact ttatccaaca atacgtacaa aatagtttca 74100 gcataacaat actaatactg ctaccaacag caaactactt agtacaactg tttttttttt 74160 ttggacattg tttttgcact tagactatag tccattaagg gtatctagtt agagttatat 74220 atatagttag atacatttgt ttctgttgat attcagtgtt aggatgtgat tttttatatt 74280 cttattctga aattctaaaa aaggtttgca tgattcaaaa cttaaaaaaa tgtggccagg 74340 catagtagct catgcctgta atcctagcaa tttgggaggc cgaggccgga ggatcccttg 74400 aatccaggag ttcgagacca gcctgggcaa caaactgaga ccctcatctc catatattaa 74460 aaaaataaaa aataaataat tgttttttta aaccacacaa aaaatgtata aacatgtatc 74520 ccctgtactg caaaatcttg ctctcattcc tattccttct gctctcttcc aaccatacct 74580 atgtagctaa ccacatttat tcattctggc tcattcttta tatgtttctt cttgcatata 74640 taagcaaact tgtatatatt taatattctt acttacagaa aatgtagtat atatactcct 74700 ttgcaccttg tatttttgtt tgtttactta agaatatatt ccagaaatca ttctacatta 74760 gttcataggc atctttttga ctattattat ggccagtttc ttaaatggct gcatagtact 74820 ctgttgtatg gttggatctt gctttagtcg accttctgta aatgggaatt attatggaga 74880 tgtagcttta gagtaaattc ctagaagaat gattgctggt caaagagtaa atgcatatgt 74940 agttttatta gatgttgtca aatttccttc catatgagca gcacacaatc acttttaaaa 75000 ggcacttttt tggccggggg tggtggctca cgcctgtaat cccagcactt tgggaggcca 75060 gggcaggagg atcatgaggt caggagatgg agactattct ggctaacagg gtgaaaccct 75120 gtctctacta aaaaatacaa aaaattagcc gggcgtgttg gcgcatgcct gtagtcccag 75180 ctactcagga ggctgaggca ggagaattgc ttgaacctgg gaggcagagg ttgcagtgag 75240 ctgagatcgc accactacgc gccaggctgg gcaatagagc gagacttcgt ctcaaaaaaa 75300 aaaaaaaaaa ggcacttttt tgatttgtac atttgctaat tatatgaatc aatctagaac 75360 tttcttctgg aacaactata gcctcttttt gcttttgtgt tttgttttgc ctctacctaa 75420 cctatttttt gaatcagttt acttttgtga attaatctca aattcttgca acctgcttgt 75480 cacttttaac aattattaaa atctttccct tcttatttta atacatagtt cataggaaag 75540 tatttcattg ccagttatga agtctacatg attgttttag ccttttattt cttcagaaag 75600 taatataaat cttacttttc tggaaggtag tattagatct atttatacat atggaggaaa 75660 atttgaggat aattgagtca ttccatcacc actatccatg agacaaagac ttttccatac 75720 caagtgtctt taaagcatat ttgtagaatt aaataaaata tagctataca tattaaaaat 75780 ctttttaaaa aacttctgga aggaaggtat tatctaattg gacatactaa gttttcagtg 75840 ctgcattttg agcaaattaa agttggtcat aaaaaatact tacctttggc tggacacggt 75900 ggctcacacc tgtaatccca gcactttggg aggccaaggt gggcggatct cgaggtcagg 75960 agatcgagac catcctggct aacacagtga aaccccatct ctactaaaaa tacagaaaat 76020 tagccaggtg cctgtagtcc cagctactcg ggaggctgag gcaggagagt ggtgtgaacc 76080 caggaggcgg agcttgcagt gagccgagat cacaccactg caccccagtc tgggcgacag 76140 agcgagagtc gtctcaaaaa aaaaaaactt accttttgga gaacacagaa caaaaacttt 76200 gtcctagaaa tagtgaaaat caaaagggga acttacattc atcagagaca actctgagat 76260 tatgtgagaa tgctattttc tttttgtcaa gaaattttgt ctactaaaca tctctaagta 76320 aatattaagt acccaacaga aaaggatgga gactgtaaca aaacatgcaa aacagaaagc 76380 cttaccttct ggaatgatct caatcaccag agaaataaca ttttaatatg atcttaacaa 76440 tttcaaccat aacgtatgtt gaacatttac actctggcag gcactgtgat aggcacgtta 76500 tctgtattct catttgattt ttaaaacaat cctccaaaaa gggagattgg tattcacatc 76560 atatgtctag ttaatattct aatgtctttc cattctaaaa tcccatggac tttgtttctc 76620 ccattagcat aggaatataa ttttaaagag actcttaata ttcaaatttt aaaacaagtt 76680 cgttttgatc actggaaata aatgcttatt ttaaaaatct ttaccatggc ttttcaaagc 76740 tatatgacta gatttgcatt ttatgggtaa ttgagatatt actttgtttt actgaaaatc 76800 ttggctctct ataactcaca gaaatctatc atttttgata ggaaaatatt ctgattttaa 76860 tctttcatgt attaacttta tgttatagga atatttctaa aagatcttct ctacttgctg 76920 ttccctataa aagacagatg tagagctaga taatatatag cacataggtt ttcatgactg 76980 tgtaacctgt gctaaagtac tcttatcacg tattctcaca catagcacat gtatatatgc 77040 attagcaagt gaacatagat gctgaatttt taaatttctt ttgttgatat ccctgcaagg 77100 gacaaactct ttttagagag aaagagcaga agtcacctga tttatctccc aggtcaatga 77160 taatgatata ggcggctttt ctgactgtag atatttttct ttttcttttt tgtctttttt 77220 tttttttttt ttttttttag tcactgctct gggtgactgc tcagggtggt ttccttaaca 77280 gaagtcttgt ttgctactca ctgcttctgt ttctttttcc aatttctgcc aacttctgca 77340 atttgctgct tgtgttatca gttctttccc tcctcttaag tcttcctaat tttcgtattt 77400 tctgtagctc cacctatgtt cctctgttgt gtgagaaatg gcagtcctac gaaatacatt 77460 ggtcagcttg tcaaactaaa aggaagagac agtgtgattg gctgacaatc atagtatttt 77520 atttgatctt attattaaaa caagcaaaaa actctgagga tactttccct gattataaaa 77580 gtaacaccca ttcattgtgt taaatttagg agatacagaa aagggaaatc acatgtaaat 77640 gatggttact ttatgatatg ttcctgtatt taaaaattat tcttattttt tggtctgtgc 77700 gtatgtactt ttagagacaa cattttgatc tactgggcat attctattgt aactctcctt 77760 tttcacttaa gaatatagtg tgtgcatctt tatatgtacc atatagtgtt tctaaaaata 77820 gattttaatt tagcaaagac tttaaaaatt tccttcaaaa tcagagttca acaaaaagtc 77880 ccaaaatttg gaaaggcgag atgttggcta gatttgcaat taatgtttgt tggctcatta 77940 taatagctga tattggctgg gcccagtggc tcatgcctgt aatcacagca ctttgggagg 78000 ccgaggcggg cggatcacga ggtcaggaga tggagaccat cctggctaac acggtgaaac 78060 ccagtctcca ctaaaaaata caaaaaatta gccgggtgtg gtgccacgcg cctgtagtcc 78120 tagcaactcg ggagactgag gcaggagaat tgcttgaacc caggacgggg aggttgcggt 78180 gagtagagat tgcactactg cactccagcc tgggcgacag agcaagactc agtctcagga 78240 aaaaaaaaaa aaaatagctg ttatctattg aatatttacc gtgtgtcaga cactgtgata 78300 agtatttatt gaaacgatct gctttgattc ttctaggatg tttgtgctca atactttatg 78360 tggattatcc aatttaattc ttagaccatg aagtaggagc tattctatta ttcacattac 78420 agtagaagaa acagaggcac attttgtccc agttcaccta gcctagttag tgacagagct 78480 agagtgaaac ccaggcagtc ttgatttcag taagcactcc tatatgcacc atgctatcct 78540 gtctcttcct aggcactggg aattgctttc catactttca ctttactttc ttgtattttg 78600 tttttaattt tagaattggt ggagtgttct ggttattgaa gtaactttaa cgaatttgta 78660 atataaaatg ttggtaaata atcataaagt attcaagaat aaatgtctta cctaaacaat 78720 ttatgttgct gtacaaatga agaatacaca tctatttttt atatatttag ggggtgtaaa 78780 tatgtaaatt catattttat actgttttct aataatttca agttatttgg tctgattttt 78840 ataagatagg actagattat cagtatttat atcttgtaaa gtacctaaca aaaaatatat 78900 tctaagagaa tctaaagtgt tctttcttca ttttcttatt tgcttatgat tgcattagta 78960 ctattaccta atataatatt atttgtaata ttattttgac tgtctttgca ggaatatatt 79020 tagaccactt tctgttttga gacgatttgt atcattgaaa ctaaacagtg tagccggaaa 79080 gggaaggagt gatggtgtca ctgccagtct gtgcctggat tcccactctg ccctcaggag 79140 accttgagaa ggggtgtgag aaatgaatac tgacatgcag accaagggcc gctaggcagt 79200 gtgtatatta attattcttg aagattagtg tatttctttt tttagattaa gaaaatgtga 79260 attcccaaaa ttgtctttct ataccttatt ttggagttca ctactataaa tttaatagca 79320 aactacactt ttcaatactc agattatctt tatcatgata tgttgaggta gatattaaaa 79380 ttaatgtctc ggccaggcac gatggctcac acctgtaatc ccaacacttt gggaggccaa 79440 gatgggcaga tcacgaggtc aagagatcga gaccatcctg gccaacatgg cgaaacctta 79500 tctctaataa atacaaaaat tagccgggcg tggtggcatg cacctgtagt cccagctact 79560 cgggaggctg aggcaggaga atctcttgaa cccaggaggt ggaggttgca gtgagccgag 79620 atggcgccac tgcactccag cctggcaaca gagtgaaact ccatctcaaa aaaaaaaaaa 79680 atatattatg tctcatgaat ttacagcaag ttttggtgag tgtaatgggg ttaaagtgac 79740 atatatgtga ttggagcttt cacgtgtatt ccttataagc tgtcctacag agtttttttc 79800 tgcaaagtca gaaagtgtaa agcaccatat gtttgcggct tctaactggc ttactgtgtt 79860 tagattgctc aattgcaaat tatatctaga gtgtgaagtg tcagtccttg tgactttcaa 79920 cttgggactg aaatttgagc ccaccattat ttttcttctt atggctccta acacttatga 79980 catatttttt cctcttggca gtcactgaga cttctgaatc cccagcttca acccctctaa 80040 agaatcgctg cccattccat cattcattca ttcaacaaac attactgacc acctactgca 80100 gtgcatgcta gatactgttc ttgatgtggt tgtagcagtg agccaaagaa gtaaaaatag 80160 agtaatagta ttttccagag tgagttacta ttttatcaaa tagaatatac ttgtgtattc 80220 ccaaatatac cttacaaatt tgatgaataa ctgtataaat gtttttgcat gatgttctaa 80280 ttttatctta tttagattta atccctccta aaaaaatctg tggattttat tgatatttct 80340 gctttatgga tgactataag taactgaccc taagttatat tgcaggtaaa tagaagagtt 80400 aggatttatg tttcgataat tcattccaaa cctaggaatt tttcactgtg cctttttcag 80460 cattctttaa atttcagttc tttagaattt atttatttat ttatttgtgt tctggcccca 80520 agtattctgc atgaagctac agtacaagat ggtattggct aagaatgcag attcttactc 80580 atattcaagt ttgttaacct aattgtactt gtttctactg taaaatgggt gtagtaacca 80640 tgtacctcac atgtttgtca tgaggctcaa aatagatgat atatatataa gaattcatat 80700 atatgaaaac atatgtatat atatgaatac atatagatgt acatatatga ggagagcaca 80760 aagagctact atttttatta tctttattgc agtgaaggca ggctgcaaca ttattggatc 80820 ataaagcaat tcagttttat ttattaatat tcaagtatta gtacccagaa tttgaaatag 80880 tctcatcact tattttcttt tttctttttt ttggggacgg agtcttgctc tgtagctcag 80940 gctggaatgc agtggtgcca tctcggctca ctgcaacctt tgcctcccag gttcaagtga 81000 ttctcctgcc tcagcttccc aagtagctga gattataggc acctgccatc acacccagct 81060 aatttttgtg tttttagtag agatgtttca ccatgttggc caagctgatc tcgaactccc 81120 aaccttcagt gattcgccta tctcagcctc ccaaagtgct ggtattacag acatgagcca 81180 ccgcacccag ccatcatcac ttattttttt ccttaaagat ctctcacttc cctgccctat 81240 ttttaagtgt aacgtattaa catttatata gttgtatgtc ttttacttaa caggtacaca 81300 atatttaaaa gtgtttaggt gaatgaattt tcaagtctct gttctttgaa ttttatttta 81360 gtgctgcagg tggagaaatt ttcagcctgt gtttacctga gttggctgaa atggtttctg 81420 aaaatgatgt tatcagactc attaaacaaa tacttgaagg agtttattat ctacatcaga 81480 ataacattgt acaccttgat ttaaaggtaa cgtaacgtga tttaaagtaa tgcaacatct 81540 gtgttcttct tctgctctta gtatgcacag ttgcaaactg gtattaacag gaaacaatgg 81600 ttggttcctg gtcttctaga ctgcactggt gtgctctttg cttacgtgtg cctatgtggc 81660 cttaatttta tattggcaga aaggagaaga gtgcatgtgt aagcagctca aattactggg 81720 ctcttaaaat gcatcattcc tgtgttctaa tcctacatta gatggaatta ttccattcca 81780 tcccactctt acaaccacat cctggacctt cttattttaa aatgctgtct atctctgaaa 81840 tgctcaattg tgtaatctcc atctctgaaa ataaatcctg ctaatcctca tgcatttgct 81900 cctattacct tcattcattt ttgtcttgag gttttcagtc ttttggcttc ttccagatcg 81960 ttcaggctct ttacccaagc cccgaacctc cagtctctta tctccatctc tcatgttgtt 82020 atcctttctc cttatccttc tttcataacc acacaaggac cttaagatct gaatcagtct 82080 gtgtctcatt ttttgagcat tagattcttc tgtttttata tggtgtcctt gcatgaccct 82140 ttggcttcta ttaccacttt aataataata ataattcaaa aatctccacc tacagcccag 82200 gcctctccct taatttccaa atcctcttac cgctttttca aaagaaaaaa ccctgtagtt 82260 ctgttttggg ccccactatg gagcaggggg acagtattta ccatctagta tccctgctat 82320 ttccccaact gataaaatat tggaaaagaa gcaacacagt aaatgccaag ataatcacgc 82380 tcccaccagc ttcctgttgg gtcattttcc tgctaacatc aacccaagta cattacatag 82440 catcacatag cccttgaagt gggatggcat ataaacctgt ggttgacaac aacagagcta 82500 attaccttta ccttttctcc tgtaataggc tctgtgcttt agtaataaac aagcatgaga 82560 ttgccccaaa gttaagcgac tgttataatt ctttgcacag attttgggag cagcaccttg 82620 ggcagagtat ggggttatga cagcagcagg ggactttgta ataggccgtg ggcctgcagt 82680 agtagtaaag caaagtcacg tggttttagg aggcaatgga tgaggtgtac tatgcccttt 82740 tattgtttaa gctaaagcca gggaacagtc agacctgggt catccagaat catcccattg 82800 tagtgaacca tttttcattt ttggaaatgt ttacgttttc attcctttaa gccttttttt 82860 gtgctgttcc ccagagcacc cttcgggtgg tctttttttt tttttttttt tttttttttt 82920 tttttttttt tgagacagag tctcgctctg tcgcccaggc tgaattgcag tggtgcgatc 82980 ttggctcact gcaagctcca cctcccgggt tcacgccatt cttctgcctc agcctcccaa 83040 gtagctggga ctacaggtgc ctgccaccac gcctgatttt tttgtatttt tagtagaggc 83100 ggggtttcac catgttagcc aggatggtct tgatctcctg acctcgtgat ctgcctacct 83160 cagcctccca aagtcctggg attacaggcg tgagccacca tgcccggcct cttttttttt 83220 ttaatttttt aatttttttt ttatttaaga gagggtctca ctatgttgca caggctactc 83280 ttgaactcct gagctcaaac gatcctccca cccctgcctc cagagtagct gagactacag 83340 gttgacacca ccgtacccag ctacccagag cacctttcta accagcatac tcttttaagt 83400 ctcagcttac gcatcatctc tactgcaaag ccttcttgga atttcttatt gacagaatga 83460 ggcagtcttt ttattactga gtattctata tcttaatctt tccatttaac ttcttttctc 83520 ttcaaaccta gaatcctttt tgggtgtgtt tttggcttta tgcatcattg aataatagct 83580 ccgcctaagt agtaagtcag taaatgtttg atgcatgact caatgaaatg tataatagct 83640 gagcaatatc ttccacttaa tatactttcc atctgagact agtatgcttt tttgtatatt 83700 acttatcatg tttttgttgt actttgttta gccacagaat atattactga gcagcatata 83760 ccctctcggg gacattaaaa tagtagattt tggaatgtct cgaaaaatag ggcatgcgtg 83820 tgaacttcgg gaaatcatgg gaacaccaga atatttaggt aagaatttcc ttttattttc 83880 tataccattt tgaaatttat taagtaaatg atacctctgt agaactatat tttagagcat 83940 gtaattcgat tcagttaaaa ggatttgatt ggttcaagta ttttgataca ttaaaacttg 84000 gttaggttaa gacacattta aatgaaatga ataaaagtat gaaacattta cagcaattca 84060 ggggacactg aatatgtaag tgtacaagtt gaatgtttgt gtataaagga tttgtacata 84120 aggaaaaaag aaatgggtaa gagggaagtt agggaaaaag agaaaaatgg aaaaagaaaa 84180 actgctgcag tgaagtggcc catgattgtc tttgaagcct ggctgttggc tagatctcac 84240 ttctatttct ggtcccaatt ccagagctgt tcacaataca gatatcttca gtttaaactt 84300 acaggaattg ttagccggcc acctcatttt tgtttattgc tgctgcttta gaatatgagg 84360 ttttgcatag ggttgtgaaa gtagggtttt gaagaagagt gccaaaaaga acctttgatt 84420 aggtaatctg ggcatattgt gtgaaatcaa caaattagat tatgcatatg aaaggttttt 84480 aagttgtatg aagcagtata taaatttgaa gtattttaac ttataatctt ttttcttttt 84540 tctgtttatt gtttatactg atgtccccat gtggacattg aaaagataca gttaaacaga 84600 acagatgcga ctcagctagg cagctgtctt ttccaaaaat aaaaagcaga gaaaattact 84660 aacttttgaa tgctaactgg ggctatagtc ctagaataaa tgaatgtaat tattggtaac 84720 acagctcaca gaagtatgtg aaaatgtagg tggtttaaca gaagctgact gtatatgatt 84780 taaaggcttt attaataaaa atgttaggct ttaggaaaca gtttctcttt ggagaaatta 84840 tgaaaccata ttgtgaaaca agaaacttat ttttcactca taaatcctag tttggactgt 84900 actaattcga aataatatgg taatgtttag aaagcataga gaatttattt tgcacttttt 84960 tttttggcaa gtgaagacag cagaaaatta attgctttgg aaattaggtc cttgataatt 85020 taagaggaat gtttaattca ctggaaaatc tagcatttct ggctgggtgc agtggctcac 85080 acttgtaatc ccagcacttt gggaggccaa ggcgagtgga tcacttgaag tcaggagttc 85140 gagaccagcc tgaccaacat ggtgaaaccc catctctact aaaaatacaa aattagccag 85200 gtgtggtggt gcatgcctgt agtcccacct acttgggagg ctgaggcagg agaatcgctt 85260 gaatccaaga ggtggaggtt gcagtgagcc aagatcatca tgccatttgt actccagcct 85320 gggcaacaag agcgaaactc cgtcagaaaa aagaaaatct agcatttccg agaagcaaaa 85380 tttgctcatg atgtcattgc agatgcacat gatcctattt accccaaaag aaaaacctaa 85440 attaatctaa gaaaataatg caaattaatt ctaaagtaat aaaactaatt ttttttttaa 85500 attctagctt ccagagtttt acttttttat aattgttttt ttctgagatt aacctaaatg 85560 agtttaacta cctagccact aattactcca agaatattat ccatgttttt gaaccacttg 85620 atcatataat ttactcaata ataggnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 85680 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 85740 nnnnntgtga tgaaaactca gttgagtaaa agaacaaaat acttgcagta cattgctaca 85800 tattttatga tgaaggtgaa atgcttaata agcatagaag cagcagcaga cagtccattg 85860 ccgggctcca cctcacaaag ggagacattt gatttaggtt taaaaaagtt ggccgggcgt 85920 ggtggcttac acctgtaatc ccagcacttt gggaggccaa ggcaggtgga tcatcaggtc 85980 aggagttaga gaccagcctg gccaacatag cagaacccta tttttactaa aaatatgaaa 86040 attagctgga cgtggtggcg cgtacctgta atcccagcta ctcaggatgc tgaggcagga 86100 gaatcacttg aaccagggag gcagaggttg cagtgagcca agatcatgct attgcactct 86160 agcctgggtg acaaaaaaaa aaaaaaaaaa aaaaagctag aagaaggtat ttgttaagca 86220 gaataattag gtcaggccca cccaaaaatg tacttttatg agagtacatc tctgggaaat 86280 tatgagtttt tgtagctagc caagagagaa taccataggg aatagaacca gaactgatac 86340 tggagcttca gttgagacca caaaggggac tgactcctca gaccaagtgg aaggtttaga 86400 cctgttctgt gttctgtgga actgctcatg ggtttgccca ggaaaaaaga tggtatggct 86460 aaataagttg gtgaaacatg tactatattg tctttttctg gctcctgggc accagacttg 86520 ccattaacac atttacagat tttaaaaggt ttgcagtgaa aaaatttgcc tgcccagctt 86580 tgtgcttcgc caaacttgca gcctctattt gtgcagaacc tctctttaca tctcacaagt 86640 cattactttg ccattggaaa ccacttagga aatgctgcag cagctttgtg tactccacgg 86700 caggtatctg tgtgaaggat agacggtagc aataatatat attgaataca ttgtatttat 86760 ataacacgtg cattcccttg tgttctcttt ctgtgaggca cactttgaga tcattatttt 86820 agttttaata atataggaac tttgaaaagc aatgtaaatc tgtagtttaa ccgtaccagt 86880 tttgaggaga ggagtatgtt gaatgtcgat tgtactttgt ataaatgttt aaaaattaac 86940 atacatttta agttcccctc ccttgttttt caggaatatt ggtataatag catatatgtt 87000 gttaactcac acatcaccat ttgtgggaga agataatcaa gaaacatacc tcaatatttc 87060 tcaagttaat gtagattatt cggaagaaac tttttcatca gtttcacagc tggccacaga 87120 ctttattcag agccttttag taaaaaatcc agagtaagta ataatttgtt taaatactat 87180 tacaaattta atttgtttaa atactattac agaataaata atatatacct attaagaaag 87240 tatattctaa aatnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 87300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnctaaaaa 87360 tacaaaaaaa ttagccaggc atggtggcag gcatctgtag tcccagctac ttgggaggct 87420 gaggcagaat ggcgtgaatc cgggaggcag agcttccagt aagcagagat cgcgccactg 87480 cactctagcc tagatgacag agcaagactc cgtctcaaaa aaataaataa aataaaataa 87540 ttttacatac ataacaagta tcatttactg attgtgaaat cttgaaggat tggatgccct 87600 ttgacaacct tacaaaagaa gcgagtcagc tgagcgaggt ggctcacgcc tgtattccca 87660 gcactttggg aggccaaggc ggccagatca tgaggtcagg agttcgagac tagcctggcc 87720 aatatagtga aacaccatct ctactaaaaa tacaaaagtt agccgggcat ggtggcatgc 87780 acctatagtt ccagctactc aggaggctga ggcaggagaa ttgcttgaac ccagaaagca 87840 gaggttgtgg tgagctgaga tcgcaccact gcactccagc ctgggtgaca gagtgagact 87900 acattgcaaa aaaaaaaacc aaaaaaaaac caaaagtgat ttggaggtta gttaagcacc 87960 gtctttcaaa atataaagac ccactagctt tactagtaac tcatcctatg aacataatta 88020 gagaagatcc caaaaaacat tttctcaata ttcattgaag catatttata atagcaaaat 88080 gctagaagct tcaatgggca tctggttaaa taaattatgg tgtatccata tggaagagta 88140 ctaaataata cagctgttaa taaatatgtt aactctgtac tgatgtggaa aaatgcttga 88200 gatgcattat tgagtaataa agcaaattgc agaaacatgg agtatgtgaa gctgtgtatg 88260 tgtatgtgtg tgtatatgtt tgtgtatgta tatgcacata tatgtagaaa aaatatctgg 88320 aagtgggatt ggggaatgag aaaacaattt tactttttat cttcatgaga gataatatgg 88380 tgtaatgctt aaaagcacag acgatagagt caaagttcca cttaactacc atttacaagt 88440 taggagattt ggggtaaatt tcaagcctcc tggtacccta ctttgcatat ctgttaaatg 88500 agggtaataa tagtacccat ttcatgagtt tgttatgatt gttaaataat gtatttgaag 88560 cgtatagaat agtccctggc atataactaa gcattcagta aatgttagca gctattatca 88620 ttattattct cttctgtact gtttggattt tatactatga gtatgtttta ttttcacaag 88680 aagtaaaaaa tatgtctata atctatcaaa ctgaagacat tttcaacttg atttttagtt 88740 ctcaattttc ccttggtgct ttctttcaaa ggaaaagacc aacagcagag atatgccttt 88800 ctcattcttg gctacagcag tgggactttg aaaacttgtt tcaccctgaa gaaacttcca 88860 gttcctctca aactcaggat cattctgtaa ggtcctctga agacaagact tctaaatcct 88920 cctgtaatgg aacctgtggt gatagagaag acaaagagaa tatcccagag gatagcagca 88980 tggtttccaa aagatttcgt ttcgatgact cattacccaa tccccatgaa cttgtttcag 89040 atttgctctg ttagcacttt tttctttgac tcatttggac tgaatttgaa attttatatc 89100 cactccagtg agattatgat ttgtagcttc atatatgaca tgtttatatt gtaaatgcac 89160 ttttccatgg aataatttag ggaagtgttt taatgttaaa ttactagttg ctagcatgtt 89220 atgatttcat atcctgagat agctctgcag ataagaaaat atttaaatat atgacaaaaa 89280 gtaaaattgt acatgtgagt ttacatgtta atgaaataat tcaacttcaa atgaacttac 89340 cagaatgttt tgcatatcaa cannnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 89400 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 89460 nnaaggtgac tcttatacca tgcctctatc aacataattt gtttaggaaa gcagtatgaa 89520 gtttaagcca aaataatttc tactttatag atgctcaaga gacattttac aattgaaaat 89580 gtctttcaat tacaaatatt ttgaaacttc gtaagatttt cattctctgt ggtctgttat 89640 atgagagaga tcctttaact agagcaaaga gggagttaga aacctgatca gggatattct 89700 ttacaagttg gagcagagga aagagtagca tgccttcgta ttttaacgca aatgtctttt 89760 tcctcctccc aacctacttg agatctgata aggtctggaa gatggagata tttggtatgc 89820 aagtgtagag ttttttaatc ctccagaatt tctagagtag aagatactta ggtatagtta 89880 aatattctgt atttttagtc aaacatattt attaattgaa tatagaagaa aatgttgaca 89940 cactcagaca gcttactgaa ttttagatgt cttctgcatc ttagaataca agccagtcat 90000 tcagagttct aaaagtatgc ataaaaaatt acagcaccgg taggtctatt aacacagtgc 90060 ccgagtcagc ggtagcaaga ctgatgtgat cataaaacat gacatcaggc tcgtctgaag 90120 ttcttgtgtg aaattcctag tgagtgagga ggctcagctt aaagccatct gcagagtggc 90180 ccctcattgt ggtcttttgc tgggaccaat gcaagagact agggagagca aaatgtttgc 90240 ttatggctag agactatatc cagccctaat gatggggaaa gttagtcctt ttcgggtaat 90300 cttttatgaa ttttcacctg atgaccgtta tattggtctg ttatcatgtt acgataactg 90360 tgatctcatg accatgttgc tgtatcagaa gaaatagttt gacaaatggt aacaacaacc 90420 tgatgttccc cctttagacc tttaacttct caaaattttg gtaagtttcc aaattcttta 90480 ataataactt aaaacttttt gaataactat caggtcactt tatttgacca catggtgaat 90540 tcctttaatg tcttcagcat ttgttaagga aaagttttct ctacttgtgt gtgtatgtgt 90600 gcacatgtgt gtatgtacag gtgtatgtat atatctatag atagatacaa tacattcttn 90660 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 90720 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnc tgtaaattcc tgcctttttt 90780 cctgatatta agttttat 90798 <210> SEQ ID NO 5 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 5 tcacgagaag ccaggtcaca 20 <210> SEQ ID NO 6 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 6 ctccgaacgt ggcaggat 18 <210> SEQ ID NO 7 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 7 ccgtcggccc ttgtctggaa aagt 24 <210> SEQ ID NO 8 <211> LENGTH: 19 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 8 gaaggtgaag gtcggagtc 19 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Primer <400> SEQUENCE: 9 gaagatggtg atgggatttc 20 <210> SEQ ID NO 10 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: PCR Probe <400> SEQUENCE: 10 caagcttccc gttctcagcc 20 <210> SEQ ID NO 11 <211> LENGTH: 1627 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (262)...(1380) <400> SEQUENCE: 11 ctccgctgct gtcgccagga gtcacttcac gagaagccag gtcacaaccg tcggcccttg 60 tctggaaaag taaaagtgga tcctgccacg ttcggagctc cctggcgcct cgcccggctg 120 gagctagaga actcgtcctg tggcggcccc cggcgtgggg cgggacagcg gccccctgga 180 gggggcagtc ccgggagaac ctgcggcggc cggagcggta aaaataagtg actaaagaag 240 cagacctggg aatcacctaa c atg tcg agg agg aga ttt gat tgc cga agt 291 Met Ser Arg Arg Arg Phe Asp Cys Arg Ser 1 5 10 att tca ggc cta cta act aca act cct caa att cca ata aaa atg gaa 339 Ile Ser Gly Leu Leu Thr Thr Thr Pro Gln Ile Pro Ile Lys Met Glu 15 20 25 aac ttt aat aat ttc tat ata ctt aca tct aaa gag cta ggg aga gga 387 Asn Phe Asn Asn Phe Tyr Ile Leu Thr Ser Lys Glu Leu Gly Arg Gly 30 35 40 aaa ttt gct gtg gtt aga caa tgt ata tca aaa tct act ggc caa gaa 435 Lys Phe Ala Val Val Arg Gln Cys Ile Ser Lys Ser Thr Gly Gln Glu 45 50 55 tat gct gca aaa ttt cta aaa aag aga aga aga gga cag gat tgt cgg 483 Tyr Ala Ala Lys Phe Leu Lys Lys Arg Arg Arg Gly Gln Asp Cys Arg 60 65 70 gca gaa att tta cac gag att gct gtg ctt gaa ttg gca aag tct tgt 531 Ala Glu Ile Leu His Glu Ile Ala Val Leu Glu Leu Ala Lys Ser Cys 75 80 85 90 ccc cgt gtt att aat ctt cat gag gtc tat gaa aat aca agt gaa atc 579 Pro Arg Val Ile Asn Leu His Glu Val Tyr Glu Asn Thr Ser Glu Ile 95 100 105 att ttg ata ttg gaa tat gct gca ggt gga gaa att ttc agc ctg tgt 627 Ile Leu Ile Leu Glu Tyr Ala Ala Gly Gly Glu Ile Phe Ser Leu Cys 110 115 120 tta cct gag ttg gct gaa atg gtt tct gaa aat gat gtt atc aga ctc 675 Leu Pro Glu Leu Ala Glu Met Val Ser Glu Asn Asp Val Ile Arg Leu 125 130 135 att aaa caa ata ctt gaa gga gtt tat tat cta cat cag aat aac att 723 Ile Lys Gln Ile Leu Glu Gly Val Tyr Tyr Leu His Gln Asn Asn Ile 140 145 150 gta cac ctt gat tta aag cca cag aat ata tta ctg agc agc ata tac 771 Val His Leu Asp Leu Lys Pro Gln Asn Ile Leu Leu Ser Ser Ile Tyr 155 160 165 170 cct ctc ggg gac att aaa ata gta gat ttt gga atg tct cga aaa ata 819 Pro Leu Gly Asp Ile Lys Ile Val Asp Phe Gly Met Ser Arg Lys Ile 175 180 185 ggg cat gcg tgt gaa ctt cgg gaa atc atg gga aca cca gaa tat tta 867 Gly His Ala Cys Glu Leu Arg Glu Ile Met Gly Thr Pro Glu Tyr Leu 190 195 200 gct cca gaa atc ctg aac tat gat ccc att acc aca gca aca gat atg 915 Ala Pro Glu Ile Leu Asn Tyr Asp Pro Ile Thr Thr Ala Thr Asp Met 205 210 215 tgg aat att ggt ata ata gca tat atg ttg tta act cac aca tca cca 963 Trp Asn Ile Gly Ile Ile Ala Tyr Met Leu Leu Thr His Thr Ser Pro 220 225 230 ttt gtg gga gaa gat aat caa gaa aca tac ctc aat att tct caa gtt 1011 Phe Val Gly Glu Asp Asn Gln Glu Thr Tyr Leu Asn Ile Ser Gln Val 235 240 245 250 aat gta gat tat tcg gaa gaa act ttt tca tca gtt tca cag ctg gcc 1059 Asn Val Asp Tyr Ser Glu Glu Thr Phe Ser Ser Val Ser Gln Leu Ala 255 260 265 aca gac ttt att cag agc ctt tta gta aaa aat cca gag aaa aga cca 1107 Thr Asp Phe Ile Gln Ser Leu Leu Val Lys Asn Pro Glu Lys Arg Pro 270 275 280 aca gca gag ata tgc ctt tct cat tct tgg cta cag cag tgg gac ttt 1155 Thr Ala Glu Ile Cys Leu Ser His Ser Trp Leu Gln Gln Trp Asp Phe 285 290 295 gaa aac ttg ttt cac cct gaa gaa act tcc agt tcc tct caa act cag 1203 Glu Asn Leu Phe His Pro Glu Glu Thr Ser Ser Ser Ser Gln Thr Gln 300 305 310 gat cat tct gta agg tcc tct gaa gac aag act tct aaa tcc tcc tgt 1251 Asp His Ser Val Arg Ser Ser Glu Asp Lys Thr Ser Lys Ser Ser Cys 315 320 325 330 aat gga acc tgt ggt gat aga gaa gac aaa gag aat atc cca gag gat 1299 Asn Gly Thr Cys Gly Asp Arg Glu Asp Lys Glu Asn Ile Pro Glu Asp 335 340 345 agc agc atg gtt tcc aaa aga ttt cgt ttc gat gac tca tta ccc aat 1347 Ser Ser Met Val Ser Lys Arg Phe Arg Phe Asp Asp Ser Leu Pro Asn 350 355 360 ccc cat gaa ctt gtt tca gat ttg ctc tgt tag cacttttttc tttgactcat 1400 Pro His Glu Leu Val Ser Asp Leu Leu Cys 365 370 ttggactgaa tttgaaattt tatatccact ccagtgagat tatgatttgt agcttcatat 1460 atgacatgtt tatattgtaa atgcactttt ccatggaata atttagggaa gtgttttaat 1520 gttaaattac tagttgctag catgttatga tttcatatcc tgagatagct ctgcagataa 1580 gaaaatattt aaatatatga caaaaagtaa aattgtacat gtgaaag 1627 <210> SEQ ID NO 12 <220> FEATURE: <400> SEQUENCE: 12 000 <210> SEQ ID NO 13 <211> LENGTH: 424 <212> TYPE: DNA <213> ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY: 3′UTR <222> LOCATION: (8)...(424) <400> SEQUENCE: 13 cacgaggtat atgacaaaaa gtaaaattgt acatgtgagt ttacatgtta atgaaataat 60 tcaacttcaa atgaacttac cagaatgttt tgcatatcaa caaaaaaagt ggcttgagtt 120 ttattatagt tggtgtaaac tgaacacagt gaagacattg gaatttaata ggttctctct 180 ctaaggtgac tcttatacca tgcctctatc aacataattt gtttaggaaa gcagtatgaa 240 gtttaagcca aaataatttc tactttatag atgctcaaga gacattttac agttgaaaat 300 gtctttcaat tacaaatatt ttgaaacttc gtaagatttt cattctctgt ggtctgttat 360 atgagagaga tcctttaact agagcaaaga gggagttaga aacctgatca gggatattct 420 ttac 424 <210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 14 agtgactcct ggcgacagca 20 <210> SEQ ID NO 15 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 15 tcaaatctcc tcctcgacat 20 <210> SEQ ID NO 16 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 16 aatcaaggtg tacaatgtta 20 <210> SEQ ID NO 17 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 17 tcacacgcat gccctatttt 20 <210> SEQ ID NO 18 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 18 gacaatcctg tcctcttctt 20 <210> SEQ ID NO 19 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 19 tgctatcctc tgggatattc 20 <210> SEQ ID NO 20 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 20 agttttcaaa gtcccactgc 20 <210> SEQ ID NO 21 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 21 cttgagaaat attgaggtat 20 <210> SEQ ID NO 22 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 22 tgtgagttaa caacatatat 20 <210> SEQ ID NO 23 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 23 caggctgaaa atttctccac 20 <210> SEQ ID NO 24 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 24 tcctgagttt gagaggaact 20 <210> SEQ ID NO 25 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 25 gatatgaaat cataacatgc 20 <210> SEQ ID NO 26 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 26 agaaaccatt tcagccaact 20 <210> SEQ ID NO 27 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 27 ggaactggaa gtttcttcag 20 <210> SEQ ID NO 28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 28 tgagaggaac tggaagtttc 20 <210> SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 29 acatcatttt cagaaaccat 20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 30 atatgctgct cagtaatata 20 <210> SEQ ID NO 31 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 31 cacagcaaat tttcctctcc 20 <210> SEQ ID NO 32 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 32 atgttaggtg attcccaggt 20 <210> SEQ ID NO 33 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 33 tcgtgaagtg actcctggcg 20 <210> SEQ ID NO 34 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 34 aaactgatga aaaagtttct 20 <210> SEQ ID NO 35 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 35 ggagtggata taaaatttca 20 <210> SEQ ID NO 36 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 36 tcaggatatg aaatcataac 20 <210> SEQ ID NO 37 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 37 atgaaatcat aacatgctag 20 <210> SEQ ID NO 38 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 38 gaagtttctt cagggtgaaa 20 <210> SEQ ID NO 39 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 39 gatttctgga gctaaatatt 20 <210> SEQ ID NO 40 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 40 tcttttggaa accatgctgc 20 <210> SEQ ID NO 41 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 41 gtcttcagag gaccttacag 20 <210> SEQ ID NO 42 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 42 tacaatgtta ttctgatgta 20 <210> SEQ ID NO 43 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 43 catttcagcc aactcaggta 20 <210> SEQ ID NO 44 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 44 aggcctgaaa tacttcggca 20 <210> SEQ ID NO 45 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 45 agacaagggc cgacggttgt 20 <210> SEQ ID NO 46 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 46 tctagctcca gccgggcgag 20 <210> SEQ ID NO 47 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 47 tgctagcaac tagtaattta 20 <210> SEQ ID NO 48 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 48 tattccacat atctgttgct 20 <210> SEQ ID NO 49 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 49 tcctcctcga catgttaggt 20 <210> SEQ ID NO 50 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 50 atgtagataa taaactcctt 20 <210> SEQ ID NO 51 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 51 cgacggttgt gacctggctt 20 <210> SEQ ID NO 52 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 52 cgaacgtggc aggatccact 20 <210> SEQ ID NO 53 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 53 aggacgagtt ctctagctcc 20 <210> SEQ ID NO 54 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 54 aggtctgctt ctttagtcac 20 <210> SEQ ID NO 55 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 55 aaattttgca gcatattctt 20 <210> SEQ ID NO 56 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 56 tgtaaaattt ctgcccgaca 20 <210> SEQ ID NO 57 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 57 acctgcagca tattccaata 20 <210> SEQ ID NO 58 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 58 actcaggtaa acacaggctg 20 <210> SEQ ID NO 59 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 59 tcaagtattt gtttaatgag 20 <210> SEQ ID NO 60 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 60 tattctgtgg ctttaaatca 20 <210> SEQ ID NO 61 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 61 ctggtgttcc catgatttcc 20 <210> SEQ ID NO 62 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 62 tcttctccca caaatggtga 20 <210> SEQ ID NO 63 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 63 agtctgtggc cagctgtgaa 20 <210> SEQ ID NO 64 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 64 gtcccactgc tgtagccaag 20 <210> SEQ ID NO 65 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 65 gttccattac aggaggattt 20 <210> SEQ ID NO 66 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 66 ctatcaccac aggttccatt 20 <210> SEQ ID NO 67 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 67 aagtgctaac agagcaaatc 20 <210> SEQ ID NO 68 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 68 catggaaaag tgcatttaca 20 <210> SEQ ID NO 69 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 69 ctaaattatt ccatggaaaa 20 <210> SEQ ID NO 70 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 70 ctttcacatg tacaatttta 20 <210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 71 ttaaagagca cattctatcc 20 <210> SEQ ID NO 72 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 72 ttatttttac ctatgcaaaa 20 <210> SEQ ID NO 73 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 73 agttagcata acctcacatt 20 <210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 74 agtgttgctc tgtcgcccag 20 <210> SEQ ID NO 75 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 75 ctactagtta tgcttgggca 20 <210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 76 gttacgttac ctttaaatca 20 <210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 77 tattctgtgg ctaaacaaag 20 <210> SEQ ID NO 78 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 78 tggtcttttc ctttgaaaga 20 <210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 79 agcttgcagt cgcggtttga 20 <210> SEQ ID NO 80 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 80 ttatttttac cgctccggcc 20 <210> SEQ ID NO 81 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 81 taaaactcaa gccacttttt 20 <210> SEQ ID NO 82 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 82 attaaattcc aatgtcttca 20 <210> SEQ ID NO 83 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 83 ggcatggtat aagagtcacc 20 <210> SEQ ID NO 84 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide <400> SEQUENCE: 84 aaagacattt tcaactgtaa 20 <210> SEQ ID NO 85 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 85 tgctgtcgcc aggagtcact 20 <210> SEQ ID NO 86 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 86 atgtcgagga ggagatttga 20 <210> SEQ ID NO 87 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 87 taacattgta caccttgatt 20 <210> SEQ ID NO 88 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 88 aaaatagggc atgcgtgtga 20 <210> SEQ ID NO 89 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 89 gaatatccca gaggatagca 20 <210> SEQ ID NO 90 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 90 atacctcaat atttctcaag 20 <210> SEQ ID NO 91 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 91 atatatgttg ttaactcaca 20 <210> SEQ ID NO 92 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 92 gtggagaaat tttcagcctg 20 <210> SEQ ID NO 93 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 93 agttcctctc aaactcagga 20 <210> SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 94 gcatgttatg atttcatatc 20 <210> SEQ ID NO 95 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 95 agttggctga aatggtttct 20 <210> SEQ ID NO 96 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 96 ctgaagaaac ttccagttcc 20 <210> SEQ ID NO 97 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 97 gaaacttcca gttcctctca 20 <210> SEQ ID NO 98 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 98 tatattactg agcagcatat 20 <210> SEQ ID NO 99 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 99 acctgggaat cacctaacat 20 <210> SEQ ID NO 100 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 100 cgccaggagt cacttcacga 20 <210> SEQ ID NO 101 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 101 gttatgattt catatcctga 20 <210> SEQ ID NO 102 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 102 gcagcatggt ttccaaaaga 20 <210> SEQ ID NO 103 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 103 tgccgaagta tttcaggcct 20 <210> SEQ ID NO 104 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 104 acaaccgtcg gcccttgtct 20 <210> SEQ ID NO 105 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 105 ctcgcccggc tggagctaga 20 <210> SEQ ID NO 106 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 106 taaattacta gttgctagca 20 <210> SEQ ID NO 107 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 107 agcaacagat atgtggaata 20 <210> SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 108 acctaacatg tcgaggagga 20 <210> SEQ ID NO 109 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 109 aagccaggtc acaaccgtcg 20 <210> SEQ ID NO 110 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 110 agtggatcct gccacgttcg 20 <210> SEQ ID NO 111 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 111 aagaatatgc tgcaaaattt 20 <210> SEQ ID NO 112 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 112 tgtcgggcag aaattttaca 20 <210> SEQ ID NO 113 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 113 tattggaata tgctgcaggt 20 <210> SEQ ID NO 114 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 114 cagcctgtgt ttacctgagt 20 <210> SEQ ID NO 115 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 115 ctcattaaac aaatacttga 20 <210> SEQ ID NO 116 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 116 tgatttaaag ccacagaata 20 <210> SEQ ID NO 117 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 117 ggaaatcatg ggaacaccag 20 <210> SEQ ID NO 118 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 118 tcaccatttg tgggagaaga 20 <210> SEQ ID NO 119 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 119 ttcacagctg gccacagact 20 <210> SEQ ID NO 120 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 120 cttggctaca gcagtgggac 20 <210> SEQ ID NO 121 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 121 aaatcctcct gtaatggaac 20 <210> SEQ ID NO 122 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 122 aatggaacct gtggtgatag 20 <210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 123 gatttgctct gttagcactt 20 <210> SEQ ID NO 124 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 124 tgtaaatgca cttttccatg 20 <210> SEQ ID NO 125 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 125 ggatagaatg tgctctttaa 20 <210> SEQ ID NO 126 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 126 ctgggcgaca gagcaacact 20 <210> SEQ ID NO 127 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 127 tgatttaaag gtaacgtaac 20 <210> SEQ ID NO 128 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 128 ctttgtttag ccacagaata 20 <210> SEQ ID NO 129 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 129 tcaaaccgcg actgcaagct 20 <210> SEQ ID NO 130 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 130 ggccggagcg gtaaaaataa 20 <210> SEQ ID NO 131 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 131 aaaaagtggc ttgagtttta 20 <210> SEQ ID NO 132 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 132 tgaagacatt ggaatttaat 20 <210> SEQ ID NO 133 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: H. sapiens <220> FEATURE: <400> SEQUENCE: 133 ggtgactctt ataccatgcc 20 

What is claimed is:
 1. A compound 8 to 80 nucleobases in length targeted to a nucleic acid molecule encoding DRAK2, wherein said compound specifically hybridizes with said nucleic acid molecule encoding DRAK2 (SEQ ID NO: 4) and inhibits the expression of DRAK2.
 2. The compound of claim 1 comprising 12 to 50 nucleobases in length.
 3. The compound of claim 2 comprising 15 to 30 nucleobases in length.
 4. The compound of claim 1 comprising an oligonucleotide.
 5. The compound of claim 4 comprising an antisense oligonucleotide.
 6. The compound of claim 4 comprising a DNA oligonucleotide.
 7. The compound of claim 4 comprising an RNA oligonucleotide.
 8. The compound of claim 4 comprising a chimeric oligonucleotide.
 9. The compound of claim 4 wherein at least a portion of said compound hybridizes with RNA to form an oligonucleotide-RNA duplex.
 10. The compound of claim 1 having at least 70% complementarity with a nucleic acid molecule encoding DRAK2 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of DRAK2.
 11. The compound of claim 1 having at least 80% complementarity with a nucleic acid molecule encoding DRAK2 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of DRAK2.
 12. The compound of claim 1 having at least 90% complementarity with a nucleic acid molecule encoding DRAK2 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of DRAK2.
 13. The compound of claim 1 having at least 95% complementarity with a nucleic acid molecule encoding DRAK2 (SEQ ID NO: 4) said compound specifically hybridizing to and inhibiting the expression of DRAK2.
 14. The compound of claim 1 having at least one modified internucleoside linkage, sugar moiety, or nucleobase.
 15. The compound of claim 1 having at least one 2′-O-methoxyethyl sugar moiety.
 16. The compound of claim 1 having at least one phosphorothioate internucleoside linkage.
 17. The compound of claim 1 having at least one 5-methylcytosine.
 18. A method of inhibiting the expression of DRAK2 in cells or tissues comprising contacting said cells or tissues with the compound of claim 1 so that expression of DRAK2 is inhibited.
 19. A method of screening for a modulator of DRAK2, the method comprising the steps of: a. contacting a preferred target segment of a nucleic acid molecule encoding DRAK2 with one or more candidate modulators of DRAK2, and b. identifying one or more modulators of DRAK2 expression which modulate the expression of DRAK2.
 20. The method of claim 19 wherein the modulator of DRAK2 expression comprises an oligonucleotide, an antisense oligonucleotide, a DNA oligonucleotide, an RNA oligonucleotide, an RNA oligonucleotide having at least a portion of said RNA oligonucleotide capable of hybridizing with RNA to form an oligonucleotide-RNA duplex, or a chimeric oligonucleotide.
 21. A diagnostic method for identifying a disease state comprising identifying the presence of DRAK2 in a sample using at least one of the primers comprising SEQ ID NOs: 5 or 6, or the probe comprising SEQ ID NO:
 7. 22. A kit or assay device comprising the compound of claim
 1. 23. A method of treating an animal having a disease or condition associated with DRAK2 comprising administering to said animal a therapeutically or prophylactically effective amount of the compound of claim 1 so that expression of DRAK2 is inhibited.
 24. The method of claim 23 wherein the disease or condition is a hyperproliferative disorder. 